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Investigations of Ultrafast Ligand Rebinding to Heme and Heme Proteins using Temperature and Strong
Magnetic Field Perturbations
A dissertation presented
by
Zhenyu Zhang
to
The Department of Physics
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in the field of
Physics
Northeastern University
Boston, Massachusetts
August 2009
2
© 2009
Zhenyu Zhang
ALL RIGHTS RESERVED
3
Investigations of Ultrafast Ligand Rebinding to Heme and Heme Proteins using Temperature and Strong
Magnetic Field Perturbations
By
Zhenyu Zhang
ABSTRACT OF DISSERTATION
Submitted in partial fulfillment of the requirements
for the Doctor of Philosophy in Physics in the Graduate School of Arts and Sciences of
Northeastern University, August 2009
4
Table of Contents
Abstract…………………………………………………………………………. 6
Acknowledgements……………………………………………………………... 10
List of Figures…………………………………………….…………………….. 11
Chapter 1: General Introduction…………………………………………...... 18
1.1 Iron & Iron-Ligand Interaction
1.2 Myoglobin and Fe-protoporphyrin IX
1.3 UV-vis Electronic Absorption Spectrum of Heme-proteins
1.4 Axial Ligands: Proximal Ligands and Distal Ligands
1.5 Time-resolved Absorption Spectroscopy of Hemoproteins
Chapter 2: Experimental Methods…………………………………………… 50
2.1 The Synchro-lock Pump-probe Laser System
2.2 Pump-probe Spectroscopy Setup for Kinetics Studies under High
Magnetic Field
2.3 Cryostat for Spinning Sample Cell with Uniform Temperature
inside as Low as 190K
2.4 Sample Preparation
2.5 Data Analysis
5
Chapter 3: Measurement of Diatomic Ligand Recombination Kinetics to Heme proteins in Strong Magnetic Fields………………………
104
3.1 Abstract
3.2 Introduction
3.3 Experimental Results
3.4 Discussion
Appendix 1: Oxygen Rebinding Kinetics to Myoglobin and its Mutants (V68W, L29W) with Temperature Variation in Aqueous Solutions and Glycerol Mixtures………………………………...
143
A1.1 Experimental Methods
A1.2 Results and Data Analysis
A1.3 Results
Appendix 2: UV-Vis Absorption Spectral Investigations of Ferrous FeII- protoporphyrin IX in Different Monomeric Complexes……...
173
Appendix 3: How does CO rebind to FeII-protoporphyrin IX? .................... 195
Appendix 4: How does NO rebind to FeII-protoporphyrin IX? .................... 206
6
Abstract
This thesis is written to summarize investigations of the mechanisms that underlie
the kinetics of diatomic ligand rebinding to the iron atom of the heme group, which is
chelated inside heme proteins. UV-vis absorption spectroscopy and ultrafast pump-probe
spectroscopy are the major investigation methods used in this thesis.
The family of heme proteins is a major object of studies for several branches of
scientific research activity. Understanding the ligand binding mechanisms and pathways
is one of the major goals for biophysics. My interests mainly focus on the physics of this
ligand binding process. Therefore, to investigate the problem, isolated from the influence
of the protein matrix, Fe-protophorphyrin IX is chosen as the prototype system in my
studies. Myoglobin, the most extensively and intensively studied protein, is another ideal
system that allows coupling the protein polypeptide matrix into the investigation.
A technique to synchro-lock two laser pulse trains electronically is applied to our
pump-probe spectroscopic studies. Based on this technique, a two color, fs/ps pump-
probe system is developed which extends the temporal window for our investigation to
13ns and fills a gap existing in previous pump-probe investigations. In order to apply this
newly-developed pump-probe laser system to implement systematic studies on the
kinetics of diatomic ligand (NO, CO, O2) rebinding to heme and heme proteins, several
experimental setups are utilized.
In Chapter 1, the essential background knowledge, which helps to understand the
iron-ligand interaction, is briefly described.
7
In Chapter 2, in addition to a description of the preparation protocols of protein
samples and details of the method for data analysis, three home-made setups are
described, which include: a picosecond laser regenerative amplifier, a pump-probe
application along the bore (2-inch in diameter) of a superconducting magnet and a
temperature-controllable cryostat for spinning sample cell.
Chapter 3 presents high magnetic field studies of several heme-ligand or protein-
ligand systems. Pump-probe spectroscopy is used to study the ligand recombination after
photolysis. No magnetic field induced rate changes are observed in any of these ligand
recombination processes within the experimental detection limit. A magnetic field
dependent CO rebinding behavior is observed for the FePPIX-CO sample in
80%glycerol/20%water environment. Careful data analysis indicates that this magnetic
field induced change is due to the amplitude difference of a “fast” (<10ps) response with
and without the magnetic field application (the amplitude changes from ~55% at 0 Tesla
to ~45% at 10 Tesla). Kinetics of CO rebinding to FePPIX in 80%glycerol at the
extremes of the magnetic field intensities (0Tesla vs. 10 Tesla) can be decomposed into a
ligand rebinding process plus two 5ps decays heme cooling with different amplitudes. It
leads to suggest a magnetic field induced change of a short-lived heme cooling response
after photolysis. Also, CO rebinding kinetics to different heme compounds demonstrates
a wide range for the Arrhenius pre-factors. This work reveals that the “spin-selection rule”
does not play a key role in the recombination process of CO to heme iron.
In Appendix 1, the recombination of oxymyoglobin and its mutants is investigated
in the temperature range from 275K to 318K, using a home-made cryostat. Quite
surprisingly, the O2 molecule rebinds to heme iron inside myoglobin with dramatically
8
different behavior as the temperature is varied, depending on the protein environment. It
shows little dependence (Mb), no dependence (V68W Mb mutant) and large dependence
(L29W Mb mutant) in this 40K temperature window. To expand this temperature
window, since the motor inside the cryostat is capable to work as low as 230K, glycerol
is introduced into the protein preparation. It is observed that protein samples in a
glycerol/water mixture, even with only 20% glycerol (in weight), the temperature
dependences of the O2 rebinding to heme iron are dramatically altered. The O2 rebinding
behavior also shows a high dependence on the glycerol concentration in the solution.
These tendencies are consistent with previous studies of other colleagues. A comparison
of kinetics in different solutions indicates that environmental viscosity must not be the
only effect inducing the glycerol-dependent behavior.
Two different methods are applied to prepare the oxymyoglobin and O2-bound
V68W myoglobin mutant. These different protocols in sample preparation also affect the
O2 rebinding kinetics.
In Appendix 2, the absorption spectra of Fe-protoporphyrin IX in different
monomeric complexes are investigated and compared. This work may suggest that, inside
the CTAB micelles, ferrous Fe-PPIX exists in an equilibrium state of different species,
CO probably can bind to FeII-PPIX with different trans ligand (H2O or OH-), and the
trans effect, exhibiting while NO binds to H93G Mb mutants might also happen when
NO binds to FeII-PPIX inside CTAB micelles.
In Appendix 3, several ultrafast kinetics studies of CO rebinding to FePPIX are
listed. They might be helpful for future studies on such systems.
9
In Appendix 4, a series of systematic studies of the NO recombination to FeIIPPIX
is performed with temperature and environment variation. A four-state model is proposed
to explain the kinetics of NO-FeIIPPIX after photolysis.
To sum-up, two scientific devices were designed, constructed and applied to
practice successfully in the protein-ligand kinetics studies. A home-made pico-second
regenerative amplifier was developed in collaboration with Prof. Anchi Yu. This laser
amplifier and the whole synchro-locked laser system was aligned successfully when
necessary. Diatomic ligands (CO, NO and O2) rebinding to ferrous heme (FeII-PPIX) or
heme proteins were investigated under various experimental schemes. Strong magnetic
fields, mutants, temperature alteration, variance of protein environment were used as
methods to perturb these protein-ligand rebinding processes.
10
Acknowledgements Foremost, I would like to express my sincere gratitude to my advisor, Dr. Paul M.
Champion. I could not have imagined a better advisor who gave me the freedom to
explore on my own, timely help with immense knowledge and simultaneously, the
guidance to recover when my exploration going astray. His patience and guidance helped
me go through the crooked road and complete this dissertation. However, his assiduity
and preciseness toward science will be cross-reference to me forever.
I would also like to thank Dr. J. Timothy Sage, whose pioneering works on bare heme
inspired me and helped me sort out the orientation of my studies. As always, a concise
discussion with Tim usually led me to key references and the breakthrough point. I owe
my deep gratitude to Prof. Dr. Armen Stepanyants for serving on my thesis committee
and his support as my reference. I must also thank Prof. Dr. Don Heiman for his
invaluable contributions to this thesis.
I would like to acknowledge Dan Sierpina, Harold Marshman and Tim Hussey for
numerous discussions and suggestions that helped me improve my knowledge in the area
of their expertise.
I am also grateful to the following current or former lab-mates: Weiqiao “Bridge” Zeng,
Dr. Abdelkrim Bennabbas, Yuhan Sun, Dr.Karunakaran Venugopal, Alexander Demidov,
Dr. Wenxiang Cao, Dr. Xiong Ye, Dr. Dan Ionascu, Dr. Flaviu Gruia, Dr. Minoru Kubo,
and Dr. Anchi Yu.
My deepest gratitude goes to my wife Xuewei, for her quiet patience, unquestioning
support and unwavering love. This thesis would be impossible without her unyielding
devotion and unending encouragement. I am indebted to our parents, whose support and
care helped us overcome setbacks and let me stay focused on my graduate study. Last but
not least, I thank our brother Xuelin and sister Yijun, who gave their full supports during
these years.
It is to my wife, my daughter and my families that this thesis is dedicated.
11
List of Figures
1.1 The shapes and orientation of the 3d orbitals……………………… 20
1.2 Partial orbital energy-level diagram for a transition-metal ion affected by ligand field………....………………………………......
21
1.3 Crystal field splitting diagrams of a transition-metal ion inside octahedral complexes………………………………………………
23
1.4 Crystal field splitting diagrams of a transition-metal ion inside other coordination complexes……………………………………...
24
1.5 The energy level scheme for ferrous iron showing the occupation of the 3d orbitals affected by strong and weak ligand fields…….....
25
1.6 Structures of oxymyoglobin…………………………………...…... 28
1.7 Electronic absorption spectra of horse heart myoglobin with different redox status, spin state and ligand ligation…………...…..
32
2.1 Conceptual arrangement of a conventional pump-probe setup…..... 51
2.2 Electronically synchrolocked pump-probe system…………….….. 53
2.3 Dual cavity optical schematic of the Coherent oscillators (Mira 900F and Mira 900P)……………………………………………….
55
2.4 Time delay calibration curve of the phase shifter for the Synchrolock-AP in the fundamental phase-locked loop…………...
58
2.5 MbNO recombination kinetics performed with optical delay and electronic delay………………………………………………….....
60
2.6 Key elements inside ultrafast pulsed laser cavities………………... 62
2.7 Construction arrangement of home-built pico-second regenerative amplifier and its real application…………………………………...
64
2.8 Timing diagram of the home-buit picosecond regenerative amplifier………………………….………………………………...
67
2.9 Absorption spectra of Glass filters used to block pump beam before the detector……………………………………………….....
69
12
2.10 Pictures of the layout for a pump-probe experiment with a cylindrical superconducting magnet…………………….……….. ..
71
2.11 Optical arrangement along the bore of the superconducting magnet 72
2.12 Pictures of the home-made cryostat for spinning sample cells…… 76
2.13 The absorption spectra of some of the heme or hemeprotein samples in deoxy and ligand-bound forms.…..………….…………
81
2.14 Convolution of a step function with a 3 picosecond Gaussian pulse 94
2.15 Convolutions of different single exponential decay processes with a 3ps Gaussian pulse………………………...……………………..
96
2.16 Two exponential decaying processes with 5% difference in time constants compared using MEM analysis and exponential simulation…………………………………………………………..
99
3.1 The rebinding kinetics of diatomic ligands (CO, NO and O2) to ferrous myoglobin at room temperature in aqueous solution……....
108
3.2 Recombination kinetics of ferric HRP-NO and MbNO with and without application of external magnetic field………………...…...
113
3.3 Comparison of NO rebinding kinetics to FeII-protoporphyrin IX with and without an applied magnetic field………………..………
115
3.4 Comparison of CO rebinding kinetics to FeII-protoporphyrin IX in 80% glycerol with and without the application of a magnetic field..
116
3.5 Detailed measurements of the magnetic field dependence kinetics of FePPIX-CO in 80% glycerol at pH=12…..……………………..
117
Table 3.1 Fitting parameters of PPIX-CO in 80% glycerol/water mixture under 0 Tesla and 10 Tesla magnetic field…………...…………….
119
3.6 Kinetics fitting with SRC model for PPIXCO under magnetic field of 0 Tesla and 10 Tesla…………......……………………………...
121
3.7 The CO rebinding kinetics to FePPIX taken with different ultrafast pump-probe laser system……...……………………………………
122
3.8 Kinetic spectral responses of PPIX-CO and deoxy PPIX in 80% glycerol/20%water solution………...………………………………
123
13
3.9 Pump-probe kinetics measurement at the ν(C-O) stretch mode wavelength……..…………………………………………………..
125
3.10 Comparison of L29WMbO2 rebinding kinetics with and without application of a magnetic field………………..……………………
126
Table 3.2 Spin changes associated with ligand binding to high-spin heme iron…………………………………………………………………
130
Table 3.3 Kinetics of CO binding to selected heme proteins…...……………. 135
A1.1 Absorption spectrum of an MbO2 sample prepared with method 1.. 144
A1.2 Absorption spectrum of an MbO2 sample prepared with method 2.. 146
A1.3 Absorption spectra comparison of horse heart MbO2 samples prepared with different methods……………..…………………….
147
A1.4 Absorption spectrum of an MbO2 sample prepared with method 2 but over-flushed with unknown amount of excess oxygen gas….....
149
A1.5 Absorption spectrum of a V68W MbO2 mutant sample prepared with method 2……...……………………………………………….
150
A1.6 Absorption spectrum of a L29W MbO2 mutant sample…...………. 151
A1.7 Temperature dependent kinetics data of horse heart MbO2 and its oxidation product MetMb……………..…………………………...
154
A1.8 Temperature dependent kinetics of MbO2 from 276K to 316K….... 156
Table A1.1 Temperature dependence of O2 rebinding to horse heart myoglobin 157
A1.9 O2 recombination kinetics to horse heart myoglobin in borate buffer at room temperature…………………………………………
158
A1.10 Glycerol concentration dependent kinetics of O2 rebinding to horse heart myoglobin at room temperature......………………………….
160
Table A1.2 Glycerol dependence of O2 rebinding to horse heart myoglobin at room temperature…..………………………………………………
161
A1.11 Temperature dependence of MbO2 rebinding kinetics inside glycerol mixture………..................………………………………..
162
A1.12 Experimental data of temperature dependent O2 rebinding kinetics to mutant V68WMb in borate buffer……………………………..
164
14
A1.13 Kinetics of O2 rebinding to V68WMb differentiated by preparation methods………………......………………………………………...
165
A1.14 Kinetics of O2 rebinding to oxymyoglobin differentiated by preparation methods…………………………………...…………...
166
A1.15 Temperature dependent recombination kinetics of horse heart MbO2 prepared with method 2……………………………..………
168
A1.16 Temperature dependent recombination kinetics of mutant V68W MbO2 inside glycerol mixture……………..……………………….
169
A1.17 Temperature dependent recombination kinetics of mutant L29W MbO2 inside borate buffer……………...…………………………..
170
A1.18 Relationship between solvent viscosity and O2 recombination kinetics to horse heart myoglobin…..……………………………...
171
Table A1.3 Viscosity of aqueous glycerol solutions………………..…………. 172
A2.1 Absorption spectrum of FeIIPPIX-CO inside Kpi buffer with 1% CTAB………..……………………………………………………..
174
A2.2 Absorption spectrum of 2MeIm-FeIIPPIX-CO inside Kpi buffer with 1% CTAB…………..………………………………………...
175
A2.3 Absorption spectrum of pyridine-FeIIPPIX-CO inside Kpi buffer with 1% CTAB…………..………………………………………...
176
A2.4 Absorption spectrum of CN-FeIIPPIX-CO inside Kpi buffer with 1% CTAB…………………..……………………………………...
177
A2.5 Absorption spectrum of phenol-FeIIPPIX-CO inside Kpi buffer with 1% CTAB……..……………………………………………...
178
A2.6 Absorption spectrum of FeIIPPIX-NO with CN- inside the Kpi buffer solution with 1% CTAB………..…………………………...
179
A2.7 Absorption spectrum of phenol-FeIIPPIX-CO inside 80% glycerol. 180
A2.8 Absorption spectrum of FeIIPPIX-CO inside 80% glycerol……….. 181
A2.9 Comparison of absorption spectra of FeIIPPIX-CO inside CTAB micelles and high concentration glycerol solution…………...…….
182
A2.10 Absorption spectrum of monohydroxy complex of FeIIPPIX…...... 183
15
A2.11 Absorption spectrum of dihydroxy complex of FeIIPPIX……….... 184
A2.12 Absorption spectral evolution of FeIIPPIX from monohydroxy complex to dihydroxy complex………...…………………………..
185
A2.13 Absorption spectral evolution of FeIIPPIX from CO- bound monohydroxy complex to dihydroxy complex…..………………...
186
A2.14 pH dependence of FeIIPPIX in Kpi buffer with 1% CTAB, indicating a monoaqua complex of FeIIPPIX………...……………
187
A2.15 Absorption spectrum of diaqua complex of FeIIPPIX……...……... 188
A2.16 Time evolution of deoxy FeIIPPIX inside basic CHES buffer with 1%CTAB (pH=9.91)…..…………………………………………...
189
A2.17 Time evolution of deoxy FeIIPPIX inside acidic MES buffer with 1%CTAB (pH=5.52)..……………………………………………...
190
A2.18 pH dependence of diaqua complex of FeIIPPIX inside 80% methanol/20% water mixture………..……………………………..
191
A2.19 Absorption spectrum of FeIIPPIX under extreme acidic environment (pH=2.3)…...…………………………………………
192
A2.20 Absorption spectrum of Ferric, Ferrous and CO- bound PPIX in 80%methanol/20%water mixture………………...………………...
193
A2.21 Absorption spectrum of Ferric, Ferrous and NO- bound PPIX in 80%methanol/20%water mixture…………………...……………...
194
A3.1 Independent responses of FeIIPPIX-CO to external magnetic field inside CTAB micelles………..…………………………………….
196
A3.2 Comparison of kinetic responses of FeIIPPIX-CO with and without 2MeIm bound as the trans ligand inside CTAB micelles……….....
197
A3.3 Pump-probe investigation on FeIIPPIX of monohydroxy complex inside CTAB micelles……..……………………………………….
198
A3.4 Pump-probe investigation on FeIIPPIX-CO inside 80%methanol/20%water mixture, probed at 435nm…………...…..
199
A3.5 Pump-probe investigation on FeIIPPIX-CO inside 80%methanol/20%water mixture, probed at 428nm………...……..
200
16
A3.6 Pump-probe investigation on 2MeIm-FeIIPPIX-CO inside 80%methanol/20%water mixture, probed at 435nm…...…………..
201
A3.7 Pump-probe investigation on 2MeIm-FeIIPPIX-CO inside 80%methanol/20%water mixture, probed at 428nm………..……..
202
A3.8 Pump-probe investigation on 2MeIm-FeIIPPIX-CO inside glycerol-CTAB-buffer ternary solution system, with variation on glycerol concentration……………………………………………...
203
A3.9 Kinetics of PPIX-CO in 80%glycerol/20%water (pH=12) compared with visible probing at 435nm and IR probing at 1955cm-1……………………………………………………………
204
A3.10 Glycerol dependent kinetics of 2MeIm-PPIX-CO in glycerol/water mixture…………..…………………………………………………
205
A4.1 NO rebinding to FeIIPPIX inside CTAB micelles with and without 2MeIm in the solution (only geminate rebinding)…..……………..
207
A4.2 NO rebinding to FeIIPPIX inside CTAB micelles with and without 2MeIm in the solution (with diatomic rebinding)……..…………...
208
A4.3 NO rebinding to FeIIPPIX inside 80%methanol/20% water mixture 209
A4.4 Comparison of NO rebinding kinetics to FeIIPPIX at room temperature with different solution environments………..………..
210
A4.5 Temperature dependent kinetics of NO rebinding to FeIIPPIX inside CTAB micelles……………………………………………...
211
A4.6 MEM analysis of temperature dependence for NO-FeIIPPIX recombination inside CTAB micelles………………..…………….
212
A4.7 Temperature dependent kinetics of NO rebinding to FeIIPPIX inside a glycerol-CTAB-buffer ternary solution…………………...
213
A4.8 Temperature dependent kinetics of NO rebinding to FeIIPPIX inside 80% glycerol/20%water mixture……………..……………..
214
A4.9 Glycerol dependent kinetics of NO rebinding to FeIIPPIX at room temperature…………………………………………………………
215
A4.10 Temperature dependent kinetics of NO rebinding to FeIIPPIX inside 80%methanol/20%water mixture…………………………...
216
17
A4.11 MEM analysis of temperature dependence for NO-FeIIPPIX inside 80%methanol/20%water mixture……………...…………………...
217
A4.12 Kinetics model proposed for NO recombination to FeIIPPIX after photolysis…………………………………………………………
218
18
Chapter 1 General Introduction
1.1 Iron & Iron-Ligand Interaction
Iron serves as an important trace element that is involved in many physical and
chemical activities of living cells and organisms(1-3). On our planet, every living
creature needs iron and it is used in a wide variety of ways. The pigeon uses iron as a
“magnet”(4) to help it travel effectively long distances to specific destinations. A recent
study(5) reveals that aggregates of magnetite nanocrystals (in Fe3+ form) located in its
upper beak tissue is the key to this ability. Also, iron is involved in strengthening the
teeth of rats (6), which are unusually strong and capable of biting through a wide variety
of materials.
Differing from those two interesting applications of iron for living creatures as
mentioned above, most of its biological functions are correlated with the redox flexibility
of iron atom and its ability to bind ligands as a transition metal ion. One of the
characteristic features of the transition metal family is that they are able to bond with a
variety of molecules or ions to build-up metal-ligand complexes through coordinate-
covalent bonds. It is worth mentioning here the difference between valence bond and
coordination bond. For the valence bond, both of the bonding atoms contribute one
unpaired electron and the chemical bondage is localized between the pair of bonding
atoms. In the case of coordination bonding, there are no localized metal-ligand bonds in
19
the complex. Ligands contribute both electrons to the coordinate-covalent bond. All the
electrons that ligands donate to the metal center are actually occupying a three-
dimensionally delocalized orbital around the central atom.
The iron atom has electronic configuration of 1s22s22p63s23p63d64s2. This is
sometimes also written as [Ar] 3d64s2. It commonly evolves in chemical reactions in two
oxidation states, or charge states, the ferrous (Fe2+) state ([Ar] 3d6) and the ferric (Fe3+)
state ([Ar] 3d5). There are five 3d orbitals (Figure1.1), therefore 10 vacancies for those 3d
electrons to occupy. For a free Fe ion, these five d orbitals are energetically identical. The
possibilities of the distribution of electrons within these orbitals are equal. When ligands
attached, the main influence on the iron atom is to break the degeneracy of the 3d orbitals.
Crystal Field Theory(7) assumes that these ligands are structureless negative charges, and
analyzes their effect on the energy levels of iron atom. Detailed description needs to be
combined with means of quantum mechanics. Hereinafter a pictorial briefing is presented
to illustrate the situation in the octahedral complex, the most common geometry in iron-
ligand interaction.
In Figure 1.2(a), to compare the crystal field effects with the 3dxy and 3dx2-y2
orbitals laying in the xy-plane, six negative charges are disposed along the Cartesian axes,
equidistantly from the origin point. On the whole, an electron in a 3dx2-y2 orbital stays
closer to these negative charges, therefore experiences more repulsion from the ligands
than does an electron in a 3dxy orbital. So, the energy of 3dx2-y2 will be higher than that of
combination of two orbitals 3dy2-z2, 3dz2-x2. It shares the same symmetry (eg symmetry)
20
3dx2‐y2 3dz2
3dxy 3dxz 3dyz
Figure 1.1 The shapes and orientation of the 3d orbitals. 3dxy 3dxz and 3dyzhave their lobes between the corresponding axes. 3dx2-y2 has its lobe along xand y axes. 3dz2 can actually be decomposed to two components: 3dy2-z2 and3dz2-x2 , which have the same shapes as the others.
21
3dxy 3dx2‐y2
_
_ _
_
_ _
_
_
Energy
Free Ion
3dx2‐y2 ,3dz2eg
3dxy ,3dyz,3dxzt2g
Δo
Figure 1.2 (a) 3dxy and 3dx2-y2orbitals orientated in the xy-plane surroundedby 6 point negative charges along the Cartesian axes equidistantly. (b)partial orbital energy-level diagram for a transition-metal ion in anoctahedron of six point negative charges. The energy of 3dx2-y2 and 3dz2 is3/5Δo higher than that of the free ion and the energy of 3dxy 3dxz 3dyz is2/5Δo lower than that the free ion.
(a)
(b)
22
with 3dx2-y2. Thus, in an octahedral complex, the 3dxy, 3dyz, 3dxy orbitals are
energetically degenerate, whose energy is lower than that of the other two degenerate
orbitals, 3dx2-y2 and 3dz2. Corresponding partial orbital energy-level diagram is listed as
Figure 1.2(b). The symbol Δo denotes the strength of the d-orbital splitting. Analogous
consideration could be applied to other ligand orientations (Figure 1.3 & Figure 1.4).
When the outer shell 6 (ferrous) or 5 (ferric) electrons of iron occupy these
splitting 3d orbitals, electron pairing energy P becomes impactive. In the electron
distribution for Fe3+, the first three electrons occupy the t2g level anyway. If Δo < P, the
fourth and fifth electrons go in the eg level, with spins parallel to those in the t2g level. It’s
the weak field, high-spin (S=5/2) case and notated as t2g3 eg
2. If Δo > P, the energy
required to overcome the repellence within the paired spin electrons won’t compete the
energy for an electron to entering the upper level. Then, the fourth and fifth electrons
locate to the t2g level and are paired with other electrons. This t2g5 eg
0 state is the strong-
field, low-spin (S=1/2) case. Figure1.5 shows the occupation of the 3d orbitals at
different strength of crystal field. Both Δ and P depend upon the iron configuration and
the ligands. A partial spectrochemical series listing ligands from small Δ to large Δ can
be attained from classical inorganic chemistry books.
1.2 Myoglobin and Fe-protoporphyrin IX
On average, there are 3 to 4 gram irons existing in the human body. 2/3 of these
irons are in hemoglobin. Another 15% of it is reserved inside ferritin, the iron-storage
23
Energy
eg
t2g
b1g (dx2‐y2)
a1g (dz2)
b2g (dxy)
eg (dxz, dyz)
b1g (dx2‐y2)
a1g (dz2)
eg (dxz, dyz)
b2g (dxy)
(a) (b) (c)
Figure 1.3 Crystal field splitting diagrams of octahedral complexes. (a) aregular octahedral complex, all six ligands are identical and equidistantlyfrom the metal ion. (b) a Jahn-Teller distorted octahedral complex, in whichthe two ligands trans to each other are further away than the other four. (c)distorted octahedral complex with the two bonds along the z-axis are shorterthan the other four in the xy plane.
24
Energy
eg
t2g
b1g (dx2‐y2)
a1g (dz2)
b2g (dxy)
eg (dxz, dyz)
b1g (dx2‐y2)
a1g (dz2)
eg (dxz, dyz)
b2g (dxy)
(a) (b) (c)
Figure 1.4 Crystal field splitting diagrams of other possible geometries ofiron-protoporphyrin complex (a) a regular octahedral complex as areference. (b) square planar complex, four coordinate (c) square pyramidalcomplex in which the metal iron is five coordinated.
25
Figure 1.5 The energy level scheme for ferrous (Fe2+) iron showing theoccupation of the 3d orbitals for strong and weak ligand fields in a distortedoctahedral geometry. On the left side, for a 5-coordinate iron, such as foundfor deoxy Mb where L1 is the proximal histidine, the electron pairing energy(P) is bigger than the cubic term (Δo) of the ligand field splitting. Under thiscondition, electrons occupy the dx2-y2 and dz2 orbitals to avoid the electronpairing energy, resulting in the high spin (S=2) state of the ferrous iron atom.On the right side, when a sixth ligand (L2) binds to the iron atom, the largecubic term in the crystal field results in pairing of the 6 d electrons and theformation of a low-spin (S=0) ferrous state. A similar situation holds forferric (Fe3+) heme complexes, leading to S=5/2 for high-spin and S=1/2 forlow-spin.
26
WEAK FIELDS = 2
STRONG FIELDS = 0
energy
d xz , d yz
d x2‐y2d z2
d xy
Free Ion
L1
L2
L1
d xz , d yz
d xy
d z2
d x2‐y2
27
Protein, for future usage. The rest is found in myoglobin, cytochromes, and other
proteins, including those needed for cell division and DNA synthesis. Hemoglobin, which
evolved about 450 millions years ago, picks up oxygen molecules from the lungs and
transports them to the tissues of the body. Each hemoglobin molecule has eight thousand
atoms elaborately weaved together(8). The primary mission of this atomic networking is
to safeguard its four ferrous iron atoms from oxidation. Once these ferrous ions are
oxidized to ferric state, they loose their capability to bind oxygen (9). Myoglobin takes
the oxygen molecules from hemoglobin and behaves as a local oxygen reservoir for the
muscle cells. These two hemeproteins share striking similarities in their structures.
Hemoglobin has four individual polypeptide units, each of which envelops a prosthetic
group identified as heme and structurally resembles the single polypeptide myoglobin.
Due to its simplicity, myoglobin is an ideal model for understanding these two oxygen-
carrier proteins(10-13).
Myoglobin (Mb) is a globular protein with a dimension of 44Åx44Åx25Å(14). It
consists of 153 amino acids, forming a single polypeptide chain, and the prosthetic heme
group (Figure 1.6(b)) with a ferrous iron (Fe2+) atom chelated inside. Amino acid
residues packed into the interior of the molecule are predominantly hydrophobic, while
the water-exposed exterior is hydrophilic. In aqueous environment, these hydrophilic
amino residues facilitate a hydration shell surrounding the myoglobin molecule(15, 16).
The polypeptide chain forms a three-dimensionally folded structure, generating several
cavities inside. The heme is embedded in the biggest cavity and divides it into the distal
and proximal pockets. A histidine (His93) inside the proximal pocket binds to the iron
and forms the only connection between the Mb polypeptide chain and the heme group.
28
(a) (b)
(c) (d)
Figure 1.6 Structure of oxymyoglobin. (a) Chemical structure. (b) Hemeprosthetic group. (c) Xenon binding sites inside the protein. (d) Distalpocket with key amino acids shown.
.
29
Figure 1.6(d) shows the crystal structure of myoglobin, focusing on the proximity
of the heme group. Other smaller cavities (for example, Xe pockets as in Figure1.6(c))
(17, 18) provide interior pathway and play important functional role for ligand
recombination(19-22) and migration(23-26). Small molecules such as oxygen (O2),
carbon monoxide (CO), and nitric oxide (NO) can reversibly dissociate from and bind to
the iron atom from the distal side of the heme plane.
Myoglobin (Mb) has been studied extensively both experimentally and
theoretically over many decades, and is often taken as a prototype of heme proteins (10,
12, 13, 27-35). Experimentally, we investigate the three main states of myoglobin:
metmyoglobin, deoxymyoglobin and ligand-bound myoglobin. In Metmyoglobin, the
iron atom exists in its ferric form (Fe3+). Both 6- and 5- coordination conformations could
exist, but normally a water molecule (H2O) is bound to the iron atom as the sixth ligand.
Other small molecules and ions such as NO, CN-, OH-, F-, N3 can also take the role, but
no situation of CO or O2 molecules binding to metmyoglobin has been observed. When it
obtains an electron from a reducing agent in aqueous solution, the iron atom reduces to
ferrous (Fe2+) form and the myoglobin turns to deoxymyoglobin, which is 5-coordinated.
At low temperature (below 100K), an intermediate state could be formed from
metmyoglobin by photo-reduction with the water molecule still bound to iron atom,
which is in the ferrous low spin state(36). In the normal situation there is no sixth ligand
in the ferrous state and the iron atom domes from the heme plane toward the proximal
histidine ligand by ~0.4Å(28) and is usually in its high spin state(S=2 for Fe2+). Diatomic
ligands (CO, NO or O2), can bind to deoxymyoglobin and pull the iron atom back to the
heme plane(37) and make it change to the low spin state (S=0).
30
Heme (Figure 1.6(a)) is iron atom chelated with protoporphyrin IX in a square
planar orientation. Protoporphyrins are tetrapyrroles containing the eight side chains: four
methyl (-CH3), two vinyl (-CH=CH2) and two propionate (-CH2-CH2-COO-). Those
side chains can be arranged in 15 ways and the number (e.g. IX) differentiates the
arrangement of these side chains. Due to its ubiquity as an active component part inside
heme protein, Fe-protoporphyrin is sometimes referred to as a model compound.
Nitrogen atoms from each of the four pyrrole rings are ligated to the iron atom through
coordination bonds. In the octahedral geometry, the axial positions are unhindered and
another two additional ligands (L1 and L2 in Figure 1.5) can also bind to the iron atom. In
myoglobin, they are histidine 93 from the polypeptide chain as L1 and a small diatomic
molecule (NO, CO or O2) for L2, respectively.
Fe3+-protoporphyrin IX can not be dissolved at low pH values(38, 39). In the
presence of aqueous alkaline solution, dimers(40) are formed. On addition of acid in the
solution (pH values decreasing), before the precipitation occurs, monomers are re-
formed(41). Electronic absorption spectrum of the Soret band varies for these two
monomer and dimer states(42). The explanation of this dimerization has been prevailed
with an oxo-bridge linking two iron centers (H2O-[Fe3+-O-Fe3+]-OH)(43). Recently,
another mechanism is proposed without the involvement of the bridging oxygen atom(44).
In the later geometry, the noncovalent π-π stacking interaction(45) from the unligated
porphyrin faces accounts for the dimerization. In this case, each of the Fe3+-
protoporphyrin IX is high spin(S=5/2), five-coordinated.
31
An effective way to break the dimerization is to encapsulate the model compound
inside aqueous micelles(46, 47). A large macromolecular cavity generated by the
aggregation of surfactant molecules in aqueous solution helps to stabilize monomeric
model hemes in various oxidation and spin states(48, 49) of the iron with different axial
ligands. On the other hand, several studies(50-53) implied that highly viscous
glycerol/water mixtures can monodisperse low concentrated model compound in alkaline
environment (54).
1.3 UV-vis Electronic Absorption Spectrum of Heme-proteins
Figure 1.7 shows the UV-vis electronic absorption spectra of myoglobin with
different redox states and spin states, as an example to illustrate the significant
differences of their expression on absorption spectrum. In the linear combination of
atomic orbitals theory(55, 56), quantitative analyses of the absorption spectral behavior
of different state should be based on a detailed molecular orbital description of the
porphyrin(56-59). Interpretation to the lowest order (60, 61) utilize a four-orbital π-
electron molecular orbitals for the porphyrin ring. While the iron porphyrin in D4h
symmetry, parts of the porphyrin electrons occupy two nearly degenerate π-binding
orbitals [a2u(π), a1u(π)]. When a photon is absorbed, electrons can be exited to the
corresponding π*-antibonding orbitals [eg(π*)], which are degenerate. These two
electronic transitions account for the Soret bands [a1u(π)→ eg(π*)] and Q bands [a2u(π)→
eg(π*)] absorptions for hemoproteins, respectively. Further coupling of π states with
vibrational energy levels results the α/β splitting of the Q band. The α-band is a (0, 0)
transition, which results from the ν=0 vibrational level of the ground π electronic state to
32
Figure 1.7 Electronic absorption spectra of horse heart myoglobin . -------metmyoglobin (Mb3+ ), 6C water molecule, as a weak ligand is on the distalside; ------- Deoxymyoglobin (Mb2+ ) 5C water molecule can’t bind toferrous myoglobin; ------- Ferrous nitric oxide myoglobin (Mb2+ NO) ; -------Ferric nitric oxide myoglobin (Mb3+ NO ).
350 400 4500.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
500 550 600 650 700
Ab
sorp
tion
(O.D
.)
X10
Wavelength (nm)
Electronic Absorption Spetra of Horse Heart Myoglobin
X25
.
33
the ν’=0 vibrational level of the π* state. The β-band results from a vibronic (0, 1)
transition.
There is another model to explain these π→π* electronic transitions in an
elegantly simple way. In this free electron molecular orbital model(62), π orbital of the
porphyrin is simplified as a one-dimensional circular orbital. Totally 18 electrons(57, 62),
one from each of the twelve carbon atoms and six from the four nitrogen atoms, occupy
the π orbital. A single electron’s energy on this circular orbital is confined by the
Schrödinger wave equation. The energy levels are . ℓ is the quantum number
for orbital angular momentum along the orbital central axis. Eighteen electrons can fill
the levels up to ℓ=±4, leaving the lowest unoccupied levels have angular momentum
number ℓ=±5. Electron promotions from the ℓ=±4 to the ℓ=±5 energy levels initiate the
Soret band (selection allowed, L=±1) and the Q band (selection forbidden, L=±9).
Similarly, the ℓ=±3 to the ℓ=±5 excitation is responsible for an N band (allowed) and
another L band (forbidden). Optical spectroscopy of hemoglobin is well explained by
taking account of this qualitative model(63).
The Soret band ranges from 360nm to 460nm and Q band is between 500nm to
600nm. The absorption around ~280nm originates from π→π* transition of the aromatic
amino acids. The RZ (Reinheitszhal) value, representing the ratio of optical density of the
Soret band to that at ~280nm (due to amino acid residues), indicates the purity of the
heme proteins. Lowering of the RZ value in the experiments sometimes is a sign of the
denaturing of the heme functional group. The band from 600nm to 800nm results from
charge-transfer transitions. Charge-transfer absorption occurs between porphyrin-iron,
34
porphyrin-axial ligands, or iron-axial ligands(63). In the infra-red region, other charge-
transfer bands exist. Water (H2O) also absorbs in the Near-IR region, so, to study other
charge-transfer bands at longer wavelengths, aqueous samples need to be dissolved in the
heavy water (D2O) to avoid the spectroscopic interference from the solvent molecules.
Electronic absorption spectra of nitride oxide (NO) binding to myoglobin of
different ligation, spin and oxidation states (Figure 1.7) could be used as a general
example to illustrate some intensity and wavelength difference of the Soret and Q bands
in different states: Reduction of the iron (Fe3+→Fe2+) causes the red-shifts of the Soret
band; for ferric state (Fe3+), in high spin (S=5/2), there is charge-transfer band at ~600-
640nm, in low spin (S=1/2), no charge absorbance in that range; for ferrous state (Fe2+),
in high spin (S=2), the Q band isn’t resolved to α and β bands, in low spin (S=0), α and β
absorbance are clearly resolved.
1.4 Axial Ligands: Proximal Ligands and Distal Ligands
The electronic state of the heme iron is modulated by the protein environment and
plays an important role in the function of a heme protein(64). Porphyrin is a strong-field
ligand that places iron close to its spin-state crossover point so that the field components
from the axial ligands exert an enormous effect on the spin states of the iron atom and
thus its behavior in heme proteins. In myoglobin, the protein donates an imidazole
nitrogen of histidine (His) as the fifth ligand to the iron atom. Different atoms can
undertake this ligation role with examples including a histidine(His) nitrogen(N) in the
peroxidases(65), a tyrosine(Tyr) oxygen(O) in catalase(66) and a cysteine(Cys) sulphur(S)
in cytochromes P450(67). In some cases, as an example of cytochromes c(68-71), a
35
electron transfer protein, it not only donates a histidine nitrogen as the fifth ligand, but
also provides a methionine(Met) sulphur from the other side, functioning as the sixth
ligation. In this arrangement, the heme group is firmly fixed on the protein polypeptide.
The iron atom is locked to six-coordinated and low spin, both in ferric and ferrous states.
Not only is the electronic state of the iron modulated by the protein environment, but also
the ligand field strength can be affected. It is not always possible to predict the strength
of a given ligand is strong or weak. Nevertheless, a roughly qualitative comparison can
be made. Typically, His, Met, Cys−, Lys, Tyr, OH-, CN-, CO and NO are strong ligands.
Weak ligands include Glu, Asp, water, F−, Cl−, Br−, and thiols (Cys−+H+).
Established by usage, these heme ligands which connect the iron atom with the
rest of the protein are designated as proximal ligands. Heme proteins that have only one
amino acid ligand can bind exogenous ligands on the distal side. The interaction of these
exogenous distal ligands with heme proteins are extensively studied to reveal the
characteristics and functions of the protein environment. Normally, neutral exogenous
ligands bind to ferrous heme more tightly than to ferric heme. Negatively charged
exogenous ligands preferentially bind to the positive charged ferric heme. Small diatomic
molecules, mostly studied of NO(72, 73), O2(28, 35, 74-76) and CO(77-79), can bind to
hemoproteins in fulfillment of their physiological functions. Myoglobin and hemoglobin
are the most widely studied heme proteins. All three diatomic molecules (NO, CO, O2)
can bind to myoglobin, and hemoglobin. Nitric oxide (NO) binds to both ferrous and
ferric myoglobin(28). Ferric myoglobin-NO has the intrinsic tendency to spontaneously
autoreduce and convert to the ferrous species(80). Also, a second NO molecule can react
with ferric NO myoglobin and generate its ferrous complex(81). Unlike nitric oxide,
36
oxygen (O2) and carbon oxide (CO) only bind to ferrous myoglobin. When O2 binds to
ferrous myoglobin(24), it pulls the proximal histidine towards the porphyrin plane and is
connected to another histidine(His-64) in the distal pocket(82, 83) through a hydrogen
bond. CO binds to the heme iron taking a linear Fe-C-O geometry(84), while NO has a
bent architecture(85, 86). Due to their electronic structure difference, NO binding can
lead to proximal ligand dissociation and form a five-coordinated NO-heme complex, but
CO prefers a six-coordinated complex retaining the ligand on the proximal side (87).
Water (H2O) and hydroxide (OH-) are also common ligands to ferric heme
proteins. In oxidized myoglobin, water binds as a weak distal ligand. In ferrous
myoglobin, water H-bonds to the histidine 64(83) in the distal pocket and plays a key role
in its affect on the distal ligation(88, 89). Water ligation is weak and its affinity for
ferrous (Fe2+) hemes is very low. Hydroxide has a negative charge on it and its affinity to
the null charge of the Fe2+-heme is low as well. Under few circumstances, water and
hydroxide ligands can bind to ferrous iron directly. A recently X-ray characterized heme,
heme ci(90, 91), brought out the observation of a water/hydroxide ligation to ferroheme
(92) iron in the protein environment of cytochromes b(6)f.
1.5 Time-resolved Absorption Spectroscopy of Hemoproteins
With the advancement of new technologies, protein-ligand interactions have been
studied by various techniques(93). Time-resolved spectroscopy is one of the powerful
tools which can monitor the short-lived intermediate states during the protein-ligand
interaction processes. These intermediate states occur transiently during the course of
reaction and may not be populated under equilibrium circumstance.
37
Flash photolysis is a method being extensively used to initiate reactions for
kinetic investigations. Q.H. Gibson is a pioneer in this field, who first applied flash
photolysis to study heme proteins(94). In the flash photolysis scheme, a sudden
perturbation results from the light applied to the protein system. Immediately after the
external perturbation, the system occupies a non-equilibrium state and then begins to
evolve back toward an equilibrium state. According Eyring’s theory, the rate constant
could be written as ∆ , where kB is the Boltzmann constant
(=1.381x10-16 ergsK-1), h is the Plank constant (=6.626x10-27 ergs) and ΔG is the Gibbs
energy of the activation. If the energy barrier of a reaction is zero (ΔG=0), at room
temperature (T=25ºC), the largest rate constant =6.25x1012 s-1. It’s a characteristic
frequency of vibrational oscillation, which needs the time resolution of the flash
photolysis to the femtosecond level. In this time range, femtosecond coherence
spectroscopy (FCS) is a unique method used to retrieve the structural and dynamic
properties of molecules by detecting transient information carried by the third order non-
linear polarizability of the molecule(31, 95). To study transition states of the protein-
ligand interaction, pico- and femto-second time resolution are needed(96).
Technically, the time resolution of the ultrafast time-resolved absorption
spectroscopy doesn’t depend on the detector’s response time but depends on the
energy/time profile of the laser pulse used in photolysis and in subsequent observation by
a spatially delayed probe pulse. It also depends on the effective rate of the system to
generate the initial photolyzed state and the accuracy of the determination of zero time
(overlap of pump and probe pulses in time). Another important concept of all the
photochemical reactions is the quantum yield. The quantum yield of a radiation-induced
38
process is the probability that a defined event occurs per photon absorbed by the system.
Thus, the quantum yield is a measurement of the photolysis efficiency.
In experiments for time-resolved absorption spectroscopy of hemoproteins, the
bond between the heme iron and distal ligand (usually NO, CO or O2) is broken by a
photolysis laser pulse (pump laser), initiating a non-equilibrium state ensemble of the
protein molecules. Thereafter, the dissociated ligands are binding back to the iron atoms.
This event is monitored by a second laser pulse (probe laser), whose light transmitting
intensity at certain wavelength is recorded as a function of time separation between the
pump and probe laser pulses.
Based on this core concept of pump-probe scheme, several systems are developed
at our lab, emphasizing on different aspects of heme protein systems.
In one laser flash photolysis experimental setup(97, 98), the pump pulse is at
532nm with pulse width of 10ns. The probe light is a cw beam and a monochromator is
used to select the probing wavelength. Probing light signals are detected by a
photomutiplier and recorded in real time by a digital oscilloscope. The repetition rate of
the pump laser is 10Hz. A seven decade time range (10ns to 100ms) can be studied using
this setup, with two temporal resolutions (10ns or 5 us).
The multi-color pump-probe experimental setup(99, 100) is used to measure the
transient absorption spectra. Here, a white light continuum pulse is used as the probe. The
pump pulse could be set at 580nm or 400nm with pulse width less than 50fs. The probe
wavelengths range from 380nm to 540nm. Two techniques of signal detection are used in
39
this design. One is using photodiode array and the other is with photomultiplier tube
combined with lock-in amplification. The former method is usually used in transient
spectra measurement because it gives the measurement with absolute optical density. And,
the latter has higher signal to noise ratio, which is ideal for single wavelength
measurement. The repetition rate of the system is 250 kHz. Samples are held inside an
anaerobic spinning cell. This spinning cell, which is 2 inch in diameter, can rotate up to
7000 rpm. According to the refresh-rate of different sample species, the spinning speed
needs to be adjusted to ensure that, upon every pump-probe pulse pair arriving, the
sample volume perturbed by the previous pulse pair is shifted outside the beam focal area
so that fresh equilibrium material is interrogated.
Femtosecond coherence spectroscopy(31, 101, 102), or FCS is used to monitor
the nuclear oscillations of the heme proteins following a “protein-quake”, which is
initiated by a femtosecond pump pulse. In this technique, the coherent oscillations appear
along with a monotonic decaying signal. To extract weak oscillations, a high signal/noise
ratio is required. So, for this system, the pump-probe pulse train is designed at repetition
rate of 76 MHz. Therefore, it has a limitation that the protein system studied in this setup
must be refreshed within 13ns. The time delay between the pump and probe is controlled
by a stepping motor with 1 micrometer step. So, the delay between pump and probe can
be controlled as multiples of 6.666fs. Detailed knowledge of this setup could be found in
other thesis of our group.
Most of the data analyzed in this thesis are collected through a two-color
electronically delayed pump-probe system(103). Detailed description will be extended in
40
next chapter. Briefly, the pump-probe pulse pair has repetition rate of 190 kHz. The
pump pulse fixed at 403nm with pulse width ~100fs and the probe pulse width is ~3ps.
The wavelength of the probe pulse can be tuned between 410nm to 450nm. The two
pump and probe pulses can be focused onto the sample collinearly or non-collinearly. In
the collinear geometry, a long-pass filter is used to eliminate the pump leakage to the
detectors. The delay between the pump and probe pulses can be electronically controlled
from 0ns to 16ns. By manually adjusting the pump-delaying circuits, the time interval
between the pump and probe pulses can be extended up to 5 µs in steps of 13.2ns. With
this system, not only kinetics of NO, but also that of O2 and CO rebinding to heme
proteins or model compound has been studied in this thesis.
41
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Chapter 2 Experiment Methods
2.1 The Synchro-lock Pump-probe Laser System
Pump-probe techniques are normally used for investigating ultrafast phenomena.
First, the sample is suddenly disturbed by the pump pulse from its equilibrium situation
and returns to its initial state or relaxes towards a new equilibrium state therewith. To
monitor this process, a probe pulse hits the sample after an adjustable delay time and its
transmission or reflection is measured. These optical properties (S) are directly compared
to the same characteristics when the sample’s non-equilibrium relaxation is not triggered.
Recording the change of these properties (ΔS) at various delay time (t) provides the
function ΔS (t). It helps to obtain information of the phenomena initiated by the pump
pulses. In kinetics studies of ligand binding to heme proteins, the pump pulse triggers the
ligand dissociation and simultaneously starts the clock that synchronizes the probe pulse.
Notice needs to be taken that the pump pulse not only triggers the ligand dissociation but
also raises the vibrational excitation of the ligand, heme and protein matrix(1-4). The
probe pulse energy is usually much weaker than the pump pulse so that the change of
optical properties can be detected without disturbing the object under study.
Figure 2.1 sketches the conceptual geometry for a transient absorption
measurement. In this arrangement, the probe comes from the same source as pump. The
pulse train from the laser is divided into two parts by a beam splitter and sent forward
51
delay line
E (t‐τ)
E (t)BS
RR
Spinning Sample
Detector
/1.23‐‐ ‐‐‐‐‐
LIAchopper
Figure 2.1 Conceptual arrangement of a pump-probe experiment setup. Theprobe pulse branch has a optical delay line to lengthen the path which theprobe light travels. Locking Amplifier (LIA), with a mechanical chopper, isused to detect the pump-induced probing variation.
52
Along different tracks. The probe beam goes to a track including a delay line which is a
device that increases or decreases the path length the light will travel. The delay line
could be a retro-reflector sitting on a motorized stepper. Moving the retro-reflector back
and forth will change the path length the probing pulse travels. It takes the light 1fs to
propagate 0.3µm. Therefore, if we want to probe the sample at a certain delay after it has
been pumped, the simple way is to move the retro-reflector backwards and lengthen the
path correspondingly. Since the optical property changes with and without the pump
perturbation are compared and measured using lock-in detection, an “ultrafast”-response
photo detector is actually not required. Each data point of the transient absorption spectra
at delay time Δt is typically averaged over many pulses.
With this optical path length delay method, the dynamic range in time is limited
by the distance that the delay line can span. For example, to investigate a system’s kinetic
response over 10ns range, the retro-reflector should move back and forth on the rail for
1.5 meter. Compared to the pump pulse, the probe pulse travels an extra 3 meter before it
hits the sample. It brings a lot of difficulties to keep the pump and probe beams
overlapped both spatially and temporally on the sample. If the probe beam is not
perfectly collimated, while the retro-reflect mirror steps along the rail, the divergence of
the input beam keeps changing. Also, any walk-off of the probe beam from overlap with
the pump beam not only decreases the signal but also distorts the data. Therefore, when
obtaining data with a long optical delay experiment, proper beam collimation and
alignment are extremely difficult(5).
One possible solution is to set the delay between pump and probe pulses
electronically. It results from a synchronization technique of two mode-locked ultrafast
53
Figure 2.2 Electronically Synchrolocked Pump-probe System. The dottedblack lines refer to electronic connections. The legend :
54
(picosecond or femtosecond) lasers(6-8). Figure 2.2 shows our electronically
synchrolocked pump-probe system which is developed based on Coherent (Coherent, Inc.
Santa Clara, CA) ultrafast lasers (Mira 900F, and Mira 900P) and its corresponding
synchronization system (Synchro-Lock 900).
Verdi V-10/V-18 lasers are solid-state diode-pumped Nd: Vandate (Nd:YVO4)
lasers. The Nd: Vandate crystal as the gain medium provides TME00 mode 1064nm
energy transition. Inside the Verdi, a LBO (Lithium Triborate, LiB3O5) crystal is used as
the doubling optics to generate a single-frequency 532nm CW laser. The powers V-10
and V-18 can provide are at 10W and 18W, respectively. These CW laser powers are
used to pump up the Ti: Sapphire crystals inside the oscillators (Mira) and regenerative
amplifiers (RegA) as the energy reservoirs for producing ultrashort laser pulses.
Mira 900 series are self-mode-locked ultrafast oscillators that use
Titanium:sapphire crystal as the gain medium. The wavelength of Mira 900 is tunable
from approximately 700nm to 1100nm with a birefringent filter (BRF). Their repetition
rate is set at 76 MHz which is defined by the laser cavity length. The mechanism referred
to modelock laser in Mira 900 is based on a spatial self-focusing effect known as Kerr
Lens effect(9-11). A brief theory of operation for the Mira 900 can be found in their
operator’s manuals. Optical schematic of the Coherent oscillators (Mira 900F and Mira
900P) is shown in Figure 2.3. CW laser from the Verdi 10 is focused (by L1) into the
Ti:Sapphire crystal. The Ti:Sapphire crystal, cut at the Brewster angle, is placed between
two concave mirrors (M3&M4). The red solid lines represent the femtosecond operation
(Mira 900F). The femtosecond laser pulses oscillate inside the laser cavity defined by the
55
OCSlit
Starter
BRF
GTITi: Al2O3
M1
M2
M3
M4
M5
P1M6
M7 M8
P2
L1
Figure 2.3 Dual cavity optical schematic of the Coherent oscillators (Mira900F and Mira 900P) . The red solid lines represent femtosecond operation.The black dashes represent picosecond operation. L: Focusing Lens; M:mirror; OC: Out Coupler; BRF: Birefringent Filter; GTI: Gires-TournoisInterferometer.
56
high reflector mirror (M6) and the output coupler (OC). The two Brewster prisms (P1 and
P2) provide for group velocity dispersion (GVD) compensation in the laser cavity and the
birefringent filter (BRF) is used to select the central carrier wavelength. The starter is an
assembly with two galvo mounted Brewster plates with butterfly geometry. The butterfly
establishes the mode-locked operation. In the picosecond configuration (Mira 900P),
which is represented by the black slashed lines, the cavity is confined by two end mirrors,
OC and GTI mirrors. The intracavity GVD compensation inside the picosecond pulse
cavity is provided by the Gires-Tournois Interferometer (GTI) mirror.
The laser beams of these two Mira’s are monitored by two 2.0GHz photodiodes
which are fiber-coupled to the Sychro-Lock 900 controller. The two Mira’s are arranged
in a master-slave configuration. The picosecond Mira 900P (the master) pulse train are
used as the frequency reference for the whole pump-probe system. The frequency and
phase differences between the master (Mira 900P) and the slave (Mira 900F) are
continuously measured and thereafter minimized by adjusting the slave Mira 900F’s
cavity. In the daily operation, the ML (mode lock) knob of Mira 900P is switched to β-
lock as this laser is self-optimized to minimize the GVD. In counterpart, the ML knob of
the Mira 900F should be set at ML because it is the slave whose laser properties need to
be regulated continuously via the synchro-lock mechanism. Three cavity length actuators
are built inside the femtosecond laser cavity to match the slave frequency to the reference
clock: a high frequency piezo-electric transducer (PZT), a low frequency galvanometer
driven delay line and a discrete step motor-driven cavity mirror translation stage. A brief
57
description of the locking sequence is given as follows: First, the photodiode signal from
the salve femtosecond Mira is fed into a frequency diplexer and split to two outputs: the
fundamental frequency and a 9th harmonic sequence. Each of these outputs is send to a
radio frequency mixer to compare with the system references which are generated with
photodiode signal of the Mira 900P. The references also include two parts: the
fundamental frequency and a 9th harmonic “comb” which is produced by a frequency
multiplier. The fundamental frequency difference is minimized through adjusting the
motorized cavity mirror stepper. Then, after the coarse frequency adjustment has been
made, the signal from the fundamental frequency mixer is used to drive the PZT-driven
mirror through a phase-locked loop. To reach the laser phase-locked status, an electronic
phase shifter in this fundamental phase-locked loop adjusts the phase offset of the slave
laser accordingly to keep the mixer output equal to zero. At the same time, a parallel loop
sends a slower control signal to the Galvonometer actuator to continuously center the DC
drive voltage on the PZT component. In a word, the Galvo loop compensates low
frequency drift in the Mira 900F. Also, a third loop using the signal from the 9th
harmonic frequency mixer can be initiated to drive the difference to zero and further
reduce the timing jitter. However, it sacrifices the possibility to automatically shift the
phase of the slave laser related to the master laser (Mira 900P). Therefore, this third
phase-locked loop is not activated routinely in our ordinary kinetics experiment. In
practice, it is detected that the phase shifter in the fundamental loop has the capability to
shift the phase equivalent to 16.5 ns time delay. Figure 2.4 is the time delay calibration
curve of the phase shifter in the fundamental phase-locked loop. It’s a key reference for
the pump-probe experiment using this electronic delay setup. Extreme care must be taken
58
0 2 4 6 8 10
02468
10121416
Rel
ativ
e tim
e de
lay
(ns)
Voltage on phase shifter (V)
Figure 2.4 Time delay calibration curve of the phase shifter in thefundamental phase-locked loop. The X-axis is the DC voltage applied on thephase shifter.
59
to measure and document this calibration curve. Independent calibration should be
performed when necessary with a fast response oscilloscope which has a time resolution
better than nanosecond. In order to check the accuracy of the calibration, the MbNO
kinetics, with electronic and optical delay, respectively, was compared in Figure 2.5. The
optical delay is implemented with a computerized stepper (Nanomotion II, CVI Melles
Griot, Inc.), which steps in 100 µm.
The laser pulses from the two oscillators (Mira 900P and Mira 900F), act as the
seed pulses, are sent into the regenerative amplifier systems to be amplified at repetition
rate of 190 kHz. The femtosecond pulsed seeds are sent to the commercial product RegA
Model 9000 for increasing the pulse energy. The picosecond seeds are amplified via a
home-made picosecond regenerative amplifier(12). The mechanism underneath both of
the two systems is the same. The following paragraphs give a full description about the
architecture of the home-build regenerative amplifier system and its timing sequence and
electronic controls.
This home-made system consists of:
• A Titanium:sapphire crystal pumped by a branch of the Verdi laser;
• A Q-switch used to prevent lasing action until a high level of inversion is
achieved in the Ti: Sapphire crystal;
• Two cavity dumpers integrated in the cavity for the reduction of the repetition rate
from 76 MHz to 190 kHz-- one for injecting a seed pulse into the cavity, the other
for ejecting out the amplified pulse.
60
-20 0 20 40 60 80 100 1200.0
0.2
0.4
0.6
0.8
1.0
MbNO kinetics
Optical Delay Electronic Delay
Δ A
Time (ps)
Figure 2.5 MbNO recombination kinetics performed with optical delay andelectronic delay. In the optical delay, the pump-probe pulses aresynchrolocked with harmonic phase shifter. Then the delay is appliedthrough a computerized stepper with 100 um resolution.
61
Figure 2.6(a) is a picture of the Ti: sapphire crystal (Ti3+:Al2O3) in different
shapes. In Ti: sapphire crystal, the metal ion (Ti3+) is doped into the sapphire (trans-
parent host). Ti is 0.1% of concentration by weight. Sapphire (monocrystalline Al2O3)
has an excellent thermal conductivity and can easily be cut into any shapes. Since it was
first introduced in 1986(13-15), Ti:sapphire lasers dominated the fields of ultrashort pulse
`durations of a femtosecond oscillator based on this Ti:sapphire gain medium are over the
10-150fs range. The shortest pulses obtained in research laboratories have durations
around 5.5 fs(16, 17). Ti3+ 3d1 electron is responsible for the lasing emission. Figure 2.6(b)
shows the Ti:sapphire absorption/emission spectra. The upper-state lifetime of
Ti:sapphire is about 3.2 μs, and its saturation power is very high. So a high-quality,
strongly focused pump beam is necessary for the Ti:sapphire crystal.
All the electro-optic devices of Q-switch and cavity dumpers used in this
amplifier are based on the acousto-optic effect. It was first predicted by Brillouin(18) in
1921 and experimentally revealed by Lucas, Biquard(19) and Debye, Sears(20). In simple
description (Figure 2.6(c)), when a radio frequency excitation is applied to the PZT
transducer, an acoustic wave is present in the medium material. The material is cut in
shape to help the build-up of an acoustic standing wave, which produce a refractive index
grating. A beam of light passing through such a refractive index grating will be partially
diffracted. The acousto-optic modulators (Q-switch & cavity dumper) spatially control
the laser beam.
The Q-switch (ON) is used to prevent spontaneous lasing in the cavity. This
allows the Ti: sapphire crystal to store energy for the amplification of an injected seed
pulse. When the Q-switch is OFF, it gives the amplified pulse a temporal window for
62
(a) (b)
(c)
Figure 2.6 Key elements inside ultrafast laser cavities. (a) Picture of Ti:Sapphire (Ti3+:Al2O3) crystal in different shapes. (b) Absorption andemission spectra of Ti: Sapphire crystal. (c) Acoustic- optic effect. Thiseffect is used to design electro-optical devices such as Q-switch and cavitydumper.
63
certain rounds of trip and preparation to be dumped out. For the cavity dumpers, while
driven by a sufficiently short RF electronic pulse, they either diffract one pulse out from
the 76 MHz pulse train into the laser cavity or eject the amplified pulse, which carries
most of the stored energy in the Ti:Sapphire crystal, outside the cavity. After ejection of
the pulse, the Q-switch is turned back on again to allow the excitation of the Ti:sapphire
crystal, preparing for the next seed pulse injected in by the cavity dumper.
Figure 2.7 shows the construction layout of the ps Ti:sapphire regenerative
amplifier. The red solid line represents the amplifier cavity. It consists of one output
coupler (M1), two high-reflectivity flat mirrors (M2, M3) and six high-reflectivity
concave mirrors (CM1-CM6). The Ti:sapphire rod is cut at Brewster angle with 5mm in
diameter and 20 mm in length. During operation, the Ti:sapphire crystal is cooled by
thermal interchanging with a NESlab reservoir whose temperature is set at 16.5°C. The
same cooler is used to cool down the Q-switch crystal. The Q-switch crystal is a Brewster
angle oriented acoustic optical quartz (NEOS N33080-25-3-BR-I). The two cavity
dumpers (CD1 &CD2) are acoustic optical fused silica Bragg cells that are 3mm in
thickness and intersect the cavity also in the Brewster’s angle. The amplifier controllers
include two radio frequency drives (pulse switch, APE, Berlin) at 16.5W for the cavity
dumpers, another rf drive for the Q-switch (NEOS Technologies, Florida) at 10W, and a
digital divider delay unit (Quantum Technology, Inc. Lake Mary, Florida) providing the
precise timing scheme for all the electro-optical components. In addition to these, a
wattmeter (Bird Electronic Corp, Cleveland, OH) is connected to the rf drive in series to
read the output, and two fast photodiodes (Electro-optics Technology, Inc. Traverse, MI)
64
Figure 2.7 (a) Construction arrangement of home-built pico-secondregenerative amplifier: Ti: Al2O3 Ti: Sapphire crystal , 20mm cylinder;M1: Output Coupler (1% at 850 nm); M2&M3: high-reflectivity flat mirror(R>99.8%); CM1-CM6: high-reflectivity concave mirror (R> 99.8%);CD1&CD2: cavity dumper, fused silica Bragg cell, 3mm; QS : Q-switch,crystal quartz. IM1&IM2: injection mirror for seed pulse. L: Convex fusedsilica focus lens (f=150mm). (b) Picture of the real application. The redsolids outline the cavity.
65
(a)
(b)
66
are applied. One photodiode is connected to an oscilloscope to monitor the amplifier
intracavity intensity, and the other one picks up the 76 MHz pulse train of the picosecond
seed laser (Mira 900P) as the external trigger for the digital divider events. All the
electronic controls are powered via an isolation transformer (1000W) to block
electromagnetic interference.
Figure 2.8 shows the timing scheme for this home-made regenerative amplifier. A
fast photodiode picks up the 76 MHz Mira 900P pulse train and sends it to the divider
delay unit (DD2). The DD2 main counter first divides the clock by 4000. After that,
synchronized to the newly- generated 190 kHz clock, three TTL signals with adjustable
timing sequence and adjustable pulse width are sent out to trigger other electronics. The
first TTL output is sent via the Out3 to the Q-switch RF driver module. The RF signal is
turned off on the rising edge of the TTL pulse for a preset duration (pulsewidth of the RF
signal). Meanwhile, the other two TTL pulses are generated and fed to the injection (out1)
and ejection (out2) cavity dumpers, respectively. These TTL signals trigger off the RF
outputs of these two driver modules. The pulsewidth of the RF output pulse is adjustable
and set as 13ns for the injection RF signal and maximum (19.8ns) for the ejection RF
signal. This arrangement ensures a whole seeding pulse could be picked up and switched
into the amplifier cavity and switched out of the cavity with the minimum residues. The
intra-cavity energy intensity can be monitored through a fast photodiode. It can be seen
that the intensity is saturated before the ejection happened. After the ejection, there is still
some residual energy left inside the cavity until the laser cavity is blocked again by the
Q-switch. The ratio of the saturated energy to the residue as well as the output energy
67
Clock Train76 MHz, 13.2ns
DD2190 kHz,5.26µs
Q-switchRF ON
Injection CavityDumper RF ON
Ejection CavityDumper RF ON
IntracavityIntensity
Amplifier Output Intensity
Out 3 Out 1 Out2
Figure 2.8 Timing Diagram of the home-built picosecond regenerativeamplifier.
68
could be used as an indication for optimization. In the pump-probe experiment for heme
proteins, the femtosecond pulses are frequency-doubled to 403nm by a BBO (β- BaB2O4)
crystal of 500µm thickness. Using a 2mm BBO crystal, the picosecond pulses are
doubled to the range 410-450nm. Together with the synchro-lock technique, they
constitute an automatic pump-probe system which has a 16ns dynamic range.
In the conventional pump-probe setup, the probe beam comes from the same
source as the pump beam. They are usually set at a non-collinear geometry. To suppress
the background light and low frequency noise of the probe, the pump is chopped before
interacting with the sample. After passing the sample, only the probe beam is sent to a
photodiode coupled with a lock-in amplifier. The pump light is blocked from reaching
the detector, in order to ensure that the pump-induced probe light change, not the pump
change itself, is measured by the lock-in amplifier. To further reject the scattered pump
light hitting the detector, the polarizations of the pump and probe pulses are set to be
orthogonal. The pump leakage contamination can be further suppressed if we use a so-
called cascaded lock-in technique(21). However, in the newly-developed system, since
the pump and probe have different wavelengths, the pump and probe beams can be
delivered to the collinearly. After interaction with the sample, the pump pulses can be
blocked by using an appropriate glass filter. In practice, when using the 403nm
femtosecond pulses as the pump, a 3mm thick long pass glass filter GG420 (Scott North
America, Inc. New York, NY) is used to filter out the pump beam. In another
arrangement, when we use the picosecond laser for pumping, and set the 403nm
69
380 385 390 395 400 405 410 415 4200
10
20
30
40
50
Tran
smis
sion
(%)
Wavelength (Nanometers)
(a)
(b)
Figure 2.9 Absorption spectra of Glass filters used to block pump beambefore the detector. (a) long pass filter to block 403nm pump pulses; (b)band pass filter to block pump pulse 435nm and let 403nm passing.
70
wavelength for probing, a band-pass filter 400FS10-25 (Andover Corp. Salem, NH) is
used to filter the pump light from reaching the detector (Figure 2.9).
2.2 Pump-probe Spectroscopy Setup for Kinetics Studies under High Magnetic Field
This setup is designed and constructed to perform pump-probe experiment while
fast spinning aqueous heme protein samples are placed in the bore of a
superconducting magnet. The closed cycle helium-cooled superconducting magnet
(CFM-14T-50) used in this work is manufactured by Cryogenic Limited (London, United
Kingdom). This superconducting magnet (Figure 2.10) has a 52mm clear room
temperature bore which is perpendicular to the top and bottom surfaces of the magnet.
The region of highly homogeneous field strength is centered over a 2mm long region at
the middle of the magnet bore. The maximum possible central field is 14.3 T with
effective field homogeneity of 0.1% in a 10mm dsv (diameter sphere volume). An upper
limit of 10T is used in these experiments. The sample interaction volume is off the bore
axis by 11mm, which leads to less than a ~1% change in the magnetic field strength
compared to the bore centerline. The magnetic field direction can be switched by
changing the direction of the superconducting current. The magnet needs ~24 hours to
cool down, prior to energizing the superconducting current. In operation, the actual
temperature near the center of the bore was 2-3 ˚C degree lower than ambient room
temperature.
The quartz sample cell is custom-made (NSG Precision Cells, Inc New York, NY)
to the specifications shown in Figure 2.11(a). The sample cell is carefully centered and
71
25”
45”
6”12.5”
(a) (b)
(c)Figure 2.10 (a) Picture of the pump-probe arrangement above the magnet.(b) Picture of the motor and it supporter underneath the magnet. (c) Designof the floor to support the magnet having level adjustability.
72
Figure 2..11 (A) The sample cell is made from quartz and the open endis typically fitted with a rubber septum cap, which is sealed withparafilm following the preparation of the sample. (B) The opticalarrangement is maintained using a cut-out aluminum tube (grey) that isinserted and coaxially aligned in the 52mm magnet bore. Lenses (B1,B2) each have a 50mm focal length and are set to focus (B1) andcollect (B2) the laser light and transfer it to the detector lens assembly(C). The sample cell (S) is glued onto a spinning shaft (E) that isconnected to a motor below the magnet. The shaft is supported andconfined by two ceramic bearings (F) on each end of a hollowaluminum cylinder (G). The larger aluminum tube insert assemblyholding the lenses is fixed to a XYZ translation stage that rests on analuminum optical table extension above the magnet. The translationstage is adjusted so that the sample resides at a point that is equidistantbetween the lenses B1 and B2. The laser pulses enter from the top, andare focused by B1 into the sample and then re-collimated by B2 so thatthey reach the detector collecting lens C. The laser pulses are thentransported by a fiber-optic cable (D) to a photodiode that is external tothe system. A long wavelength-pass filter is inserted in front of thedetector to extinguish the 403nm pump pulse. There is only 3.2mm ofclearance within the hole of the lens holding plates (B2 and C) and thespinning shaft. This is designed to be smaller than the distance betweenthe outer edge of the sample cell and the magnet bore wall. This helpsto protect the rapidly spinning sample cell from touching any surfaceand breaking.
73
Ф = 38.1mm
1±0.01mm
Central point of the magnet bore
A
A
B1
B2
C
DE
F
Ф = 52mm
101.6mm
22.3 mm
G
(a) (b)
74
glued onto an adapter which is locked onto a central shaft that spins at 3500 rpm. In order
to minimize field-induced changes in optical geometry, all the components inside the
magnet bore are designed using non-magnetic materials. For example, ceramic bearings
are supported inside an aluminum tube and used to define the position of a high-speed
spinning shaft made from hard-coat anodized aluminum. This assembly is connected to a
42V DC motor (Maxon Precision Motors, Inc. Fall River, MA) that is secured to the floor
below the magnet (Figure 2.10). The assembly holding the lenses is also made from
aluminum and it is supported by the optical table and is suspended into the magnet bore
from above. Figure 2.11(b) illustrates the geometry inside the magnet bore. In this
arrangement, the optical and mechanical parts are totally decoupled to prevent
mechanical vibrations from being transferred to the optical part of the system. The
spinning shaft could be released from its attachment to the motor below and the entire
apparatus could be pulled out of the magnet from above, in order to disconnect the optical
cell and change samples. The focal sample volume is 11.1mm away from the central axis
of the magnet bore. At such a position, the strength and homogeneity of the magnetic
field are better than 99% of their values on axis.
The incident light (pump and probe pulses) comes from above and passes through
normal focusing optics (B1) to minimize pulse distortion and deterioration of the time
resolution. Fiber optics is used to collect the light after it passes through the sample,
since the time resolved transmission has already been established at that point. The
collection lenses and fiber optic components are supplied by Thorlabs, Inc. (Newton, NJ).
The fiber is optimized at blue wavelengths and the probe laser light (435nm, 3ps, 190
KHz) is reduced in power to 80% of its original value after 2m of fiber. Fiber optic
75
collimation/coupling packages (Thorlabs, Inc. Newton, NJ) are used on both ends of the
fiber. The package (collecting lenses and mounts) are aligned in the lab and glued in
place to optimize the collimation and recovery of the ~435nm probe wavelength.
2.3 Cryostat for spinning sample cell with uniform temperature inside as low as
190K
This cryostat is designed and home-made as a supplement in the lab to the
commercial cryostat CCS-150 (Janis Research, Wilmington, MA). This Janis cryostat can
achieve temperature over the range from 14K to 300K in the experimental applications.
However, this cryostat requires a stationary sample cell and this limitation highly affects
the refreshment of the sample during the experiment. Therefore, only nitric oxide (NO)
rebinding kinetics is performed inside this cryostat because NO molecules normally have
a fast enough recombination to heme proteins. Previous works at room temperature
demonstrate that a 2-inch spinning cell (up to 7000 rpm) can refresh the sample
efficiently in our pump-probe geometry and guarantee that each pump-probe pair
interacts with an assembly of protein molecules with the identical condition. Here, a new
cryostat is constructed to hold this fast spinning cell at controllable ambient temperature.
It is tested to hold the temperature over the range of 190K to 315K non-loaded with the
stability to ±0.5K. Limited by the thermal specification of the custom-made motor
(Maxon Precision Motors, Fall River, MA) working inside the cryostat, pump-probe
experiments can be performed over the temperature range from 235K to 315K.
The dimension of the cryostat (Figure 2.12(a)) is 12inx7.5inx12in. The inner
chamber holding the uniform temperature is 9.75inx5.125inx10in. The cryostat is made
76
Figure 2.12 Pictures of the home-made cryostat for spinning sample cells.
(a) (b)
(c)
(d) (e)
(f)
(g)
77
of polycarbonate, a material which works at low temperature (up to -135 °C) and is easily
machined, molded and thermoformed. Polycarbonate parts can be tightly glued together
by using solvents whose main ingredient is chloroform (trichloromethane). The cryostat
can be decoupled into two parts: a 2.5 inch high concave base and a hood of 9.5 inch
depth. The bottom plate of the concave base is a 1 inch polycarbonate plate sandwiched
¼ inch thick Cryogel Z (Aspen aerogels, Northborough, MA). The walls of this concave
base are 1¼” high and ¾” in thickness. Two barbed polycarbonate tube connectors
penetrating one side of the walls are designed to couple the coolant circulation tubes in
and outside of the cryostat. Two methods are applied here to boost the thermal isolation
of the cryostat. First, the walls of the hood (Figure 2.12(b)) are built with double layers of
¼” polycarbonate board enveloping another 1/4” vacuum layer inside. This vacuum will
enhance the thermo-isolation of the chamber as well as prevent condensation on the glass
windows. Second, the same Cryogel Z sandwiched plates are used to form another
thermal isolation layer clung to the interior walls the hood. 1-inch holes are drilled on two
opposite walls of the hood. For each of the hole, two glass windows are mounted gas-
tightly from opposite surfaces to prevent vacuum leakage.
Inside the cryostat, there are a fan, a tube-fin heat exchanger and a home-made
motor holder (Figure 2.12(c)). The fan (MD24Z1QDN, Comair Rotron Inc, San Diego,
CA) is home-reconstructed for working under low temperature. The motor holder is made
of copper, a good thermo-conducting material. Four penstocks are drilled through the
base plate of the motor holder. Polycarbonate pipe adaptors are threaded into these
penstocks on both ends. Soft plastic tubing, which is low-temperature resistant, connects
78
adaptors on the cryostat wall, on the copper plate and to the heat exchanger. Liquid
coolant, which is pumped by an outside cooler, circulates inside the close loop and makes
heat exchange with the air inside the cryostat (Figure 2.12(d)). The whole assembly sits
on two teflon blocks which are secured to the cryostat’s bottom plate.
18 electric wires pass through the bottom of the cryostat via two gastight adaptors.
8 of these wires are used for the Maxon motor, 2 for the fan, another six lines are used
with three thermistors (ON-950-44004, Omega Engineering, Stamford, CT), two for each.
The other two are reserved for future use. All the electric wires and their controls are
compiled together inside a metal box to prevent electromagnetic perturbation from
outside (Figure 2.12 (e)). The locations of these three temperature sensors are arranged as
follows: one is dangling in the air, another one is fixed on the motor holder, and the third
is immersed into a small bottle of pure glycerol. Temperatures which are measured by
these thermistors are read out by an 866C thermometer (Omega Engineering, Stamford,
CT). To prevent condensation or frosting under low temperature on the cell, air inside the
chamber is purged with nitrogen gas before experiment. Two check valves (Figure 2.12
(f)) on the top of the cryostat are designed for this purpose. A soft gasket is placed
between the hood and the concave base to prevent air exchange within and outside the
cryostat chamber while it is working.
Protocol to run a pump-probe experiment with this home-made cryostat is
described hereinafter:
• Assemble the sample cell on the motor.
• Turn on the controls for the motor and the fan to check that they are working fine,
and then switch them off.
79
• Put on the chamber hood, switch on the motor at the necessary speed, maximize
the experimental signal by adjusting the related optics, and then turn off the motor.
• Use the six clamps to lock the hood tightly on the cryostat base, make sure the
gasket is on its right position.
• Feed nitrogen gas into the cryostat for 15 minutes, both check valves need to be
open, one for gas in, the other for gas out.
• At the same time, connect the vacuum valve to a vacuum pump and start to
vacuum the cryostat walls, turn off the vacuum valve once a rough vacuum
(0.1~10Torr) is achieved.
• Turn on the fan’s control, a small vibration can be felt by touching the cryostat.
Then turn on the motor, and switch on the cooler, setting the temperature at the
required value.
• Wait until all the three readings from the thermistors evolve to within 1°C
difference; give some extra time for the sample to be thermally equilibrated
before taking the measurement.
An important but not fully discussed issue in this thesis in the aspect of pump-
probe experiment is the concept of “magic angle” between pump-probe pulses. In viscous
solvent(e.g. Glycerol/water mixture), rotational diffusion of the hemoproteins affects the
ligand recombination kinetics(22-24). In order to eliminate this effect in kinetics studies,
one convenient way is to set the polarization directions of the pump and probe beams at
an angle of 54.7° (magic angle). For all the kinetics studies performed in this thesis, the
polarization of pump and probe pulses are always set at “magic angle”, no matter whether
the protein samples are caged in viscous solvents or non-viscous solvents. An explicit
investigation of this rotational diffusion effect on MbCO kinetics is performed by other
colleagues(12). To realize this “magic angle” geometry, a half-wave plate is first placed
along one of the laser beams (pump or probe beam) and rotated to reach its maximum
80
transmittance. After that, the wave plate is rotated for an extra 54.7° and then placed
along the other laser beam to confine its polarization. This definition of polarization
geometry is kept for all the setups constructed in this thesis.
2.4 Sample Preparation
Sample preparation is the first and the most important step for the heme-protein
kinetics studies. A subtle difference in the preparation could lead to a totally different
sample response in the pump-probe kinetics investigations. To explain the kinetics data
well and truly, one must first prepare and understand the sample under investigation. The
importance is second to none for making the right sample for the pump-probe
investigation. Figure 2.13 shows the absorption spectra of some of the samples
investigated in this study. For all the samples studied in this thesis, an elaborate
description of sample preparation will be recorded hereinafter for further reference and
comparison.
Stock Solution. Heme proteins and heme compounds usually are in ferric form
and can be purchased as commercial products in powder. Here in this thesis, horse heart
myoglobin (MetMb) and horseradish peroxidase (HRP, type VI-A) are purchased from
Sigma Chemical Co. (St. Louis, MO). Hemin (Ferriprotoporphyrin IX chloride) is
purchased from Porphyrin Products Inc. These samples are used to prepare stock
solution without further purification. Unless specially specified, all the samples are made
into extremely high-concentrated stock solutions with corresponding buffers before
further treatment. Then the stock solution is centrifuged (6000 rpm, 20°C) about 20
81
400 500 6000.0
0.5
1.0
1.5 Ferrous MbNO L29WMbO2 Deoxy Mb
400 500 6000.0
0.5
1.0
1.5
Ferric HRP-NO Ferric HRP
400 500 6000.0
0.5
1.0
1.5
O.D
. O
.D.
O.D
.
Fe(II)PPIX-CO Deoxy Fe(II)-PPIX
(in 80% glycerol)
O.D
.
400 500 6000.0
0.5
1.0
1.5
Fe(II)PPIX-NO Deoxy Fe(II)PPIX
(in 80% glycerol)
400 500 6000.0
0.5
1.0
1.5
Fe(II)PPIX-CO Deoxy Fe(II)-PPIX
(in Kpi with 1% CTAB)
O.D
.
Wavelength (nm)400 500 600
0.0
0.5
1.0
1.5
Fe(II)PPIX-NO Deoxy Fe(II)-PPIX
(in Kpi with 1% CTAB)
O.D
.
Wavelength (nm)
Figure 2.13 The absorption spectra of some of the samples investigated inthis thesis.
82
minutes longer to centrifugate possible unsolved aggregates inside with a refrigerated
centrifuge (Sorvall RT6000B, Dupont).
Normally, two types of buffer are used in the heme proteins studies here:
potassium phosphate (Kpi, pH 7.0, 100mM) and Borate (pH ~8.4, 100mM). Potassium
phosphate is used to prepare all the HRP stock and MetMb in some of the cases. Borate
buffer is used to dissolve MetMb powder for later reducing and oxygen (O2) binding
because high pH value of the environment will help to stabilize the oxymyoglobin(25).
Stock solution of hemin (FeIII-PPIX-Cl-) could be prepared in two ways. In one
way, protohemin chloride dissolved in aqueous NaOH solution (1M) is used(26). In the
other way, extra amount of solid protohemin chloride powder is added directly to 1%
(w/v) cetyltrimethylammonium bromide (CTAB) solution, and the resulting mixture then
is allowed to equilibrate inside a water bath at 50 °C for 1 hour(27). The CTAB solution
needs to be freshly made on every experimental day by dissolving CTAB powder in de-
ionized water or pH-fixed buffer.
Stock solutions of Mb mutant V68W and L29W are provided by Professor John
Olson at Rice University. The concentration of V68W mutant (ferric, Fe3+) is 1.0mM and
that of L29W mutant (ferrous, oxygen bound) is 1.2mM. When making oxygen-bound
final samples, borate buffer is used to dilute these mutants.
Sodium Dithionite Solution. Sodium dithionite (Na2S2O4) functions as a
powerful reducing agent in aqueous solutions. It is a white powder and readily soluble in
water. Usually the sodium dithionite solution is 1M and only several micro liters of
83
solution are needed from heme reduction per experiment. The following procedure is
used to generate a 1M aqueous solution of sodium dithionite.
1. Fill a 5mL glass cylindrical vial with either 1mL de-ionized water or 1mL buffer.
Put a little bit more than 0.1741 gram of sodium dithionite inside a smaller plastic
vessel and let the vessel sit on the bottom of the glass vial. The wall of the vessel
is higher than the liquid level to prevent the immediate mixing of the sodium
dithionite with the liquid.
2. Seal the vial with a septum cap and parafilm (American National Can, Menasha,
WI). Remove any residual oxygen by 3-4 rounds of vacuum-degassing followed
by flushing with argon. This can be achieved by attaching small-bore (~16 gage)
needles, to the vacuum line and argon supply, and letting them switch back and
forth to deliver the vacuum and argon through the septum seal.
3. Flip the plastic vessel and shake well to dissolve the sodium dithionite powder in
the buffer. Then, 1M sodium dithionite solution is ready for use. Sodium
dithionite solution prepared in this way can live for 8-10 hours with the high
capability of reduction.
Sodium dithionite (Na2S2O4) is a powerful reduction agent which can grab the
oxygen atoms very actively. During its storage, it gradually snatches the oxygen from the
air or even from humidity and some of it oxidized to sodium metabisulfite (Na2S2O5).
The sodium metabisulfite still has the ability to grab another oxygen and transform to
sodium dithionate (Na2S2O6). Once it’s fully oxidized to sodium dithionate, it can not
reduce ferric iron any more, but a side effect of lowering the pH value of the solution
exists. Due to this oxidization, it’s very important to seal the sodium dithionite against the
84
air. Sodium dithionate will decompose in hot water and in acid solutions(28) and its days
as iron reducer are over once decomposed.
The chemical reaction, in which sodium dithionite reduces the ferric iron atom
(Fe3+) to ferrous form (Fe2+) in aqueous solution, can be written as following formula:
2 2 2 2 4 2
Sodium dithionite also could react with another chemical sodium nitrite (NaNO2)
in aqueous solution to form nitric oxide (NO) molecules.
2 2 2 4
Nitric oxide (NO) molecules prepared in this way could be used to produce
nitrosyl myoglobin (MbNO) or other NO-bound heme proteins or model compounds.
In practice, the preparation of sodium dithionite solution could be simplified as
the following: first, half fill de-ionized water or buffer in a small glass vial (1mL) and
vacuum-degas the vial followed by flushing argon as previous described. Then, put
excess sodium dithionite powder (maybe metabisulfite/dithionate contaminated) directly
in a syringe with a small-bore needle. Use this syringe to suck water or buffer from and
then inject them back to the glass vial. When using this method, since the concentration
of sodium dithionite solution can not be estimated accurately, experience is needed to
prepare protein samples with this dithionite solution. Also, UV-Vis absorption spectra are
used to monitor if the volume of dithionite solution is appropriately applied to the sample.
The absorption of active sodium dithionite peaks around 315nm. Usually, we carefully
add the sodium dithionite solution to the heme protein samples in the way that the peak of
85
dithionite at 315nm is about the same height as the Soret peak (~400nm-440nm) of the
reduced heme proteins.
Ascorbic Acid. Ascorbic Acid (Vitamin C) may also be used as a reducing agent
(electron donor) for preparing oxymyoglobin. Ascorbate reacts with metmyoglobin
much slower than sodium dithionite does. While preparing oxymyoglobin, there is no
need to degas the metmyoglobin solution. In the open air, a small amount of ascorbic
acid powder is added to metMb solution and after 2 hours they completely react. Extreme
care must be taken because ascorbate does not only interact with metmyoglobin but also
interacts with oxymyoglobin, inducing the oxyMb→MetMb (Fe2+O2→Fe3+)
transition(29). So, it’s forbidden to shake the sample solution during the reaction. After
two hours of interaction, usually two layers of different colors could be seen of the
sample. Color of heme proteins are mainly characterized by the absorption of the Q band.
OxyMb has two sharp peaks in the Q band: one at 543nm (the green portion of the
spectrum), one at 581nm (blue portion), which gives it a bright red color. In MetMb, the
peak is shifted to 505nm in the blue portion of the spectrum and a weaker peak at 628nm
in the red portion and, this two combined together give a brown-red color. Therefore,
only the top layer of oxymyoglobin needs delivering to the sample cell for following
experiment.
Sodium Nitrite Solution and Nitric Oxide Gas. Either sodium nitrite (NaNO2)
or nitric oxide gas (NO) is used for generating NO-bound heme proteins. 1M Sodium
nitrite solution needs first to be degassed, following the same procedure described above
for sodium dithionite solution. After reducing the heme iron with sodium dithionite,
sodium nitrite is added to react with dithionite, generating NO molecules. In some cases,
86
NO-bound heme proteins are made by flushing NO gas to degassed samples. In this
situation, NO gas (99.0% purity, Med-tech, Medford, MA) is bubbled through degassed
1M sodium hydroxide (NaOH) to remove trace quantities of N2O3 and N2O4 that always
accompany in commercial NO gas product. These contaminants, if not removed, will
lower the pH value of solution and pollute it with NO2− and NO3
− ions. After that, the
NO gas is bubbled through a degassed buffer before introduced to the degassed sample.
In water, NO reacts with oxygen and water to form HNO2. Therefore, it’s very important
to ensure all the solutions the NO gas reacts with must be degassed and oxygen free.
HRP3+-NO. Dilute the stock solution of ferric HRP to around 1 O.D. /mm at the
Soret peak. Degas the diluted sample solution. Administer NO gas for 20 seconds at
pressure of 1 atm.
MbCO. Stock solution is diluted and degassed as described above. Sodium
dithionite is then added to the MetMb sample in the ratio of 1:150 (v/v) to generate the
reduced species. Then, CO gas (Med-Tech, Medford, MA) is flushed slowly over the
surface of the deoxyMb for about 30 minutes. When MbCO is prepared inside high
concentrated glycerol/water mixture, more time is needed for CO flushing until the
absorption spectrum taken by the spectrophotometer (Hitachi U-4100) indicates that the
transition is completed.
MbNO. The volume of sodium dithionite needs to be doubled because extra
dithionite is needed to generate NO molecules.
MbO2. Three ways are used to prepare oxymyoglobin in this thesis. The first way
involves ascorbic acid as described before. In the second way, oxymyoglobin is prepared
87
by adding small amount of 1M sodium dithionite solution to 1 O.D. /mm diluted MetMb
solution in the ratio of 1:100 (v/v). This results in an immediate color change as the iron
atoms reduced to ferrous form. Separate the deoxymyoglobin from the excess sodium
dithionite by passage through a sephadex G25 column that has been pre-equilibrated with
borate buffer. Once separated from dithionite, reduced myoglobin picks up oxygen from
solution to form the oxy derivative. In the third way, especially for MbO2 samples in
glycerol/water mixture, a pure oxygen supply is used. First, degas and reduce the
Metmyoglobin into deoxyMb. Then, administer pure oxygen to the sample very shortly
(< 5 seconds). Shake the solution thoroughly to have the extra sodium dithionite react
with oxygen gas. Once the sodium dithionite in the sample solution is used up, the
oxygen molecules in the solution will bind to the ferrous deoxyMb to generate oxyMb.
Finally, bring the pressure inside down to 1 atm by piercing the seal with a small-bore
needle.
Fe2+-PPIX. NO and CO binding to ferrous protoporphyrin IX are studied in this
thesis. Four environments are provided to the monomeric Fe-PPIX molecules: high-
concentrated glycerol/water mixture(30), Kpi buffer with 1% (v/v) CTAB micelles(31),
80% methanol/20% water mixture(32) and a glycerol-CTAB-water ternary system(33).
In CTAB micelles, 2-Methylimidazole (2MeIm), cyanide (CN-), Pyridine (Py) and
Phenol can ligate to the Fe2+-PPIX. CO molecule can bind to these proximal ligand
bound Fe2+-PPIX inside CTAB micelles. To prepare these samples, powder or solution of
these “proximal ligands” were added to the micelle-encapsulated Fe-PPIX solution before
degassing and reducing. The CO gas is flushed on the surface of the sample solution for
30 minutes as in the preparation for MbCO.
88
2.5 Data Analysis
Measurement of the Pump-probe Absorption Spectroscopy.
In time-resolved absorption spectroscopy experiment, the absorptions of the probe
light with and without the pump excitation are compared. The pump induced absorption
change (∆A) of the probe light are measured as
∆ ,,
Here, and are the detected probe photon densities with pump light on and off.
In practice, the geometries of the optics are arranged to ensure that, on the focal
plane, the probe focusing area is totally included inside the pump excited area. Also, the
pump and probe light intensities are controlled inside the linear-response range for the
sample. Under these conditions, the induced absorption change (over the active volume)
∆A is (34)
∆ , ∆ ,
where ∆ is the difference of the extinction coefficients of the photoproduct and reactant,
1 10 is the total absorbed pump light intensity during the
pump excitation process and Y0 is the quantum yield of the photo-excitation. Here,
is the incident pump light intensity covering the probe area, Apump is the sample
absorbance at the pump wavelength.
∆A could be simplified as
89
∆ , ∆ ,
, ,
In a simple pump-probe process, ∞
, reactant hemeprotein HL’s
are photolyzed immediately into H’s and L’s. Then, after waiting long enough, all the
ligands L’s rebind back to H’s. The system returns to its original unperturbed state, HL.
This process can be verified by comparing absorption spectra before and after the pump-
probe experiment. At time t, the photoproduct includes hemoproteins partially in ligated
state (HL), partially in un-ligated state (H). Suppose N0 is the number of reactant
molecules. NHL(t) is the number of photoproduct in HL state and NH(t) is the number of
photoproduct in H state, at time t. Letting , represent extinction coefficient per
molecule for HL and H, could be written as and
. Normally, Ligand L does not absorb at the probing wavelength
and is neglected. . Now, ∆ , could be derived as follows,
∆ , , ,
, , ,
, , ,
= , – , –
For this type of two-state processes, three characteristics could be summarized
through these equations: (1) if there is an extinction coefficient difference between the
90
reactant and photoproduct, the absorption change measurement ∆A is proportional to the
time dependent population of the photoproduct. This proportionality is independent to the
probing wavelength λ. (2) at certain wavelength λ, the larger the extinction coefficient
difference between the reactant and photoproduct, the bigger the absorption change ∆A.
(3) at certain time t, positions of extrema in the transient spectrum should overlap with
the extreme value positions on the equilibrium difference spectra – between
the reactant and product.
Now, let us consider a pseudo two-state process,
′ ′ ′ ∞′.
In this process, not only the ligated heme protein HL, but also a different ligand
L’, which is readily to bind unligated heme protein H, exists within the system for
investigation. Once the hemeprotein HL is photodissociated to H and ligand L, the other
ligand L’ can bind to unligated H first. Then, as time evolves, the original ligand L comes
back and replaces ligand L’. After long enough, all the ligand L’s will bind back to H’s
and the system returns back to its starting situation. In this type of processes, the
equilibrium absorption spectra before and after the experiment are identical and have the
same behavior as the two-state pump-probe process. So, this process is easily but
mistakenly to be treated as a two-state process described before if only the absorption
spectrum is compared.
At time t, the photoproduct includes hemoproteins partially in ligated states (HL’
and HL), partially in un-ligated state (H). Suppose N0 is the number of reactant molecules.
NHL’(t) and NHL(t) are the numbers of photoproduct in HL’ and HL states and NH(t) is the
91
number of photoproduct in H state, at time t. Letting ′ , , represent extinction
coefficient per molecule for HL’, HL and H, could be written as
and ′ ′ . Also in here, we have
′ .
Now, ∆ , could be derived as follows,
∆ , , ,
′ ′, , , ,
′ ′, , ,
′ ,
′ ′, , , ,
From this equation, the three characteristics described above for two-state
recombination do not hold. However, at the isosbestic wavelength, where ′, , ,
or at a wavelength where both HL and HL’ are silent, still can be detected because
under those two conditions, ΔA is proportional to .
In an extreme situation, let us suppose that L’ binds to H much faster than L
replaces L’. At time t1, all the unligated H’s are bound by a ligand L’. The ligand L will
not replace L’ until time t2, and finally all L’ will be replaced by ligand L. Here, t1<<t2.
Under this simplification,
(1) When t<t1, NHL=0, NHL’(t)+NH(t)=N0.
92
∆ , ′ ′, – , , – ,
′, – , , – ,
′, – , , – ′,
In this early stage, ∆A isn’t proportional to NH in general unless the experiment
wavelength λ is set at the isosbestic point of ligated species HL’ and HL. However, the
derivative,
∆ , , – ′, . It gives information of the rate
distribution.
(2) When t1<t<t2, NHL=0, NH=0, NHL’=N0.
∆ , ′, – ,
It’s a constant which is wavelength dependent.
(3) When t>t2, NH=0, NHL(t)+NHL’(t)=N0.
∆ , ′ ′, – ,
Starting from a later time t2, ∆A represents the population change of transient species
HL’. In other words, the ligand (L’) recombination process is measured hereafter.
The conceptual derivation given here can not be used to analyze real experimental
data. First, ligand L might compete with ligand L’ to rebind H in its geminate rebinding
process. Second, the process L’ rebinding to H might be entangled with the L’→L ligand
93
replacement and can not be separated temporally. And, HL’ is an intermediate state
whose equilibrium absorption spectrum ( ′, sometime is impossible to obtain.
Time Zero Defining: Numerical Simulation Study.
In the conventional pump-probe system, pump and probe pulses come from the
same laser source. Time zero can be defined by generating cross-correlation coherent
spike or with other nonlinear optical effects. There are several significant advantages of
performing the pump-probe experiments with an electronically delayed laser system.
However, in the synchrolock-delayed system, the pump and probe pulses are generated
from different source and each has different bandwidth. Therefore, it is difficult to use
non-linear effects to determine the temporal overlap of the two pulses.
Empirically, we use the experimental signal itself to define the temporal
crossing point of the two pulses, which is necessary to define time-zero in the pump-
probe experiments. In our situation, the 200fs pulse is used as the pump pulse. Because
the probe pulse is ~3ps in duration, we assume that the instrument function reacts to the
pump as an effective δ-function excitation. The observed signal is generated from the
convolution of the protein reaction and the probe pulse system response. The latter is
represented by the line shape of the 3ps (FWHM) probe laser pulse. During the initial rise
of the signal, as the pump and probe pulses overlap, the point where the signal equals 50%
of its maximum value can be assigned to time-zero to a good approximation. Figure 2.14
shows the convolution simulation of a 3ps Gaussian function with a step function which
simulates a pump-induced state jump at time zero. In this ideal situation, the step function
is used to mimic a protein reaction without decaying. It demonstrates that when the pump
94
-10 0 10 20 30 40 50
0.0
0.2
0.4
0.6
0.8
1.0
starting around 6ps the difference <1%
Temporal Overlapping
Step function Gaussian function (3ps) Convolution
Con
volu
tion
Time (ps)
Figure 2.14 Convolution of a step function with a 3ps Gaussian pulse:simulation for defining the time zero in two color pump-probe experiment.
95
and probe pulses temporally cross each other (t=0), the convolution signal equals half of
its maximum value. This feature is used to set time zero in the experiment. Routinely, in
the experiment, we first optimize the spatial overlap of the pump and probe pulses and
get the maximum of the response signal along the time axis. Second, move the
fundamental phase shift setting to position where the lock-in amplifier reading is half of
the maximum value it reached in the first step. Then, start the automatic data recording
process with the awareness that the “probe before pump” signal should be around zero.
Figure 2.15 displays convolutions of several different exponentials with a 3ps
Gaussian that simulates the probe pulse component of the instrument function.
Exponential fits using t=0 located at the half-height of the initial signal rise agree quite
well with the input time-constants (τ), so long as τ is longer than ~10ps. When τ=5ps, the
method overestimates the true time-constant by ~20%. The green lines indicate
Gaussian probe pulses of 3ps in pulse width. The blue lines show exponential decay
processes of 5ps, 10ps, 100ps and 1000ps. The small circles represent the convolved
signals that would be experimentally observed under these conditions. The convolved
signals are located along the time axis so that time-zero is fixed at the point where the
rising signal equals one-half of its maximum value. The red lines are the fits to the
convolved signals starting at the time that corresponds to the maximum value of the
signal. The data are renormalized to account for “missing amplitude” by scaling the
maximum value of the convolved signal to match the exponential decay function at its
respective time point (this function equals unity at t=0). The extracted time constants
generated by such a fitting procedure were found to be larger by 1.1ps (22%), 0.2ps
(2.0%), 0.74ps (0.7%), and 3ps (0.3%), respectively. This demonstrates that use of the
96
-10 0 10 20 30 40 500.0
0.5
1.0
τ true = 5 psτ fit =6.1 ps
N(t)
-10 0 10 20 30 40 500.0
0.5
1.0
τ true = 10 psτ fit =10.2 ps
-10 0 10 20 30 40 500.0
0.5
1.0
τ true = 100 psτ fit =100.74 ps
N(t)
Time (ps)-10 0 10 20 30 40 50
0.0
0.5
1.0
τ true =1000 psτ fit =1003 ps
Time(ps)
Figure 2.15 Convolutions of different single exponential decay processeswith a 3ps Gaussian pulse. It shows that use of the half-height of the risingsignal to define t=0 works very well when the decay is longer than 10ps.
97
half-height of the rising signal to define t=0 works very well when the decay is longer
than 10ps and overestimates the time constant by ~1ps as the decay time approaches 5ps.
Maximum Entropy Method
Most of the kinetic data analysis is analyzed with multi-exponential fitting or
maximum entropy method (MEM)(35-37). In kinetic analysis, the MEM assumes the
measurement is incomplete and inexact, so
,
where Ie is the experimental data, If is the fitting function and N(tk) is the noise at given
time. Note either If(tk) or N(tk) is unobservable. And, the noise N(tk) is assumed to be
zero-mean Gaussian with variance σk2.
, in the time domain, could be written as
∞
With the Laplace transformation, it could be written in discrete logarithmic space
like,
∆
Under the constraint,
1
98
The entropy, constructed as
ln 1
to be maximized for the fj’s. Here, Fj’s are the so called prior distribution. They are
representatives of any previous understandings that we may obtain about the rate
distribution.
Totally, there are N equations (j=1 to j=N) available for maximization of the
constructed entropy ( 0 1,2,3, … ). Iteratively solving the N equations, a set
of fj’s, constitutes a distribution of rates in logarithmic rate space, can be obtained.
Performing a numerical inverse Laplace transformation with this set, , not , is
recovered. It is an optimized kinetic trace, within some reasonable value of chi-square,
which has a minimally structured spectrum of frequencies for the kinetics studied.
The MEM approach makes minimal pre-assumptions concerning the kinetic
evolution, and yields a kinetic rate distribution that is only based on the experimental data.
However, it is worth knowing that MEM analysis can generate artifactual distributions in
the presence of incomplete (truncated) data sets and it also relies on an accurate
knowledge of the signal/noise in order to prevent underfitting or overfitting of the data.
Multi-exponential fittings are carried out with Origin (Ver 6.0, Northampton,
MA). Two single exponential decays, with 1% amplitude random noises, are analyzed
and compared on Figure 2.16. It shows that, with 1% system noise level, 5% geminate
kinetic rate constant change could be differentiated both by MEM and Origin.
99
0 200 400 600 800 1000
0.0
0.2
0.4
0.6
0.8
1.0
y=e-t/105Data:
Chi^2 = 0.00003R^2 = 0.99932 y0 -0.00074 ±0.00021A1 1.04158 ±0.00111t1 105.20779 ±0.17604
y=e-t/100Data:
Chi^2 = 0.00003R^2 = 0.9993 y0 -0.00078 ±0.00021A1 1.03682 ±0.00109t1 100.33669 ±0.16891
Val
ue
time
109 1010 10110
1
2
3
Figure 2.16 Two exponential decaying processes with 5% difference in timeconstants are compared with analysis of MEM and exponential simulation.The exponential processes are added 1% random noise on top of the data.Both MEM analysis and Origin analysis can differentiate them clearly.
100
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Chapter 3 Measurement of Diatomic Ligand Recombination
Kinetics to Heme Proteins in Strong Magnetic Fields 3.1 Abstract
Heme cooling signals and diatomic ligand recombination kinetics are measured in
strong magnetic fields (up to 10 Tesla). We examined diatomic ligand recombination to
heme model compounds (NO and CO), myoglobin (NO and O2), and horseradish
peroxidase (NO). No magnetic field induced rate changes in any of the samples were
observed within the experimental detection limit. However, in the case of CO binding to
heme in glycerol and O2 binding to myoglobin, we observe a small magnetic field
dependent change in the early time amplitude of the optical response that is assigned to
heme cooling. One possibility, consistent with this observation, is that there is a weak
magnetic field dependence of the non-radiative branching ratio into the vibrationally hot
electronic ground state during CO photolysis. Ancillary studies of the “spin-forbidden”
CO binding reaction in a variety of heme compounds in the absence of magnetic field
demonstrate a surprisingly wide range for the Arrhenius prefactor. We conclude that CO
binding to heme is not always retarded by unfavorable spin selection rules involving a
double spin-flip superexchange mechanism. In fact, it appears that the small prefactor
(~109s-1) found for CO rebinding to Mb may be anomalous, rather than the general rule
for heme-CO rebinding. These results point to unresolved fundamental issues that
underlie the theory of heme-ligand photolysis and rebinding.
105
3.2 Introduction
Iron is a crucial trace metal that is involved in many physical and chemical
activities of living cells and organisms. In one important example, it is chelated by
protoporphyrin IX (PPIX) to form the heme group. Heme proteins play key roles in
oxygen metabolism, both for oxygen and electron transport (e.g., hemoglobin, myoglobin,
cytochrome c), and as the terminal oxidase in mitochondria (cytochrome oxidase).
Recently, it has also become apparent that many heme proteins are also involved in
important regulatory processes that utilize the binding of diatomic ligands as both
catalytic and signaling agents(1-5) For this reason, it is important to understand the
mechanism of diatomic ligand binding to heme systems and to determine, for example, if
“spin selection”(6-10) rules or entropy production timescales(11) are important in
regulating this process.
When ionized, the ferrous (Fe2+) iron atom has an outer shell electronic
configuration of 3d6. In octahedral coordination complexes, such as the heme, the five-
fold d-orbital degeneracy is lifted by the ligand field of the porphyrin nitrogens and the
axial ligands. When both the axial ligands are bound, the iron d-electrons are paired in an
S=0 ground state. When one of the axial ligand is absent, the weaker ligand field leads to
a S=2 ground state(12-14). Thus, for a spin-zero ligand like CO, there is reason to
believe that the ΔS=2 nature of the reaction may require binding via a double spin flip
superexchange mechanism that slows the reaction rate and manifests itself in
significantly reduced Arrhenius prefactors, as observed for MbCO(6-8, 15).
106
In this study we investigate the effects of large magnetic fields on the ligand
binding kinetics and non-radiative relaxation of heme in glycerol solution as well as
when bound to both Mb and horseradish peroxidase (HRP). HRP catalyzes the reduction
of H2O2 to H2O in two serial one electron steps and oxidizes various aromatic substrates
in the process. HRP can bind NO in either its ferrous or ferric state and the fast geminate
kinetics of NO rebinding to this system have been reported under ambient conditions(16).
The search for magnetic field effects on diatomic ligand binding to heme in the absence
of protein material focuses on ferrous iron protoporphyrin IX (FePPIX) in 80% glycerol
solution, in the absence of 2-methylimidazole (2MeIm), so that water or hydroxide is the
likely fifth axial ligand.
Flash photolysis is an effective way to initialize ligand dissociation and study the
protein and ligand responses over many timescales(9, 17-23). During photolysis, the iron-
ligand bond is broken nearly instantaneously (probably within a fraction of the ~60fs Fe-
Ligand vibrational period(24-26)). In Mb, the Fe2+ ion moves towards the His93 and out
of the porphyrin plane by about 0.4 Å. Subsequently, the photodissociated ligand, L2,
first located inside the distal pocket, can rebind to the iron (geminate rebinding) or
explore other pockets inside the protein(27) and ultimately escape to the solvent through
protein fluctuations that involve the distal histidine(28-30). The diatomic ligands (O2, CO
and NO) undergo very different dynamical processes when rebinding to the heme in
Mb(9, 31-33). Using ultrafast pump-probe spectroscopy, it is now possible to measure the
photolysis quantum yields (QY) for these ligands(34) as well as to follow the optical
response over a very large dynamic range in time(9, 11, 19, 20, 22, 23, 26, 32, 33, 35, 36).
For heme systems, the optical response between 400-450nm includes both the ligand
107
rebinding kinetics as well as signals due to the spectral diffusion of the hot Soret band
lineshape as it undergoes vibrational relaxation back to thermal equilibrium(37-41).
Figure 3.1 shows a log-log plot of the “survival fraction”, N(t), of the deoxy Mb
photoproduct, following photolysis of NO, O2, and CO, over the time range 3ps-10ms.
One striking observation is that the three ligands have vastly different geminate rebinding
yields. At room temperature, only about 4% of the CO molecules find their way back to
the iron and rebind geminately(32, 42, 43). Thus, most of the CO molecules escape from
Mb following dissociation. In contrast, NO rebinds almost completely via ~10-100ps
geminate processes that have been analyzed intensively in several studies(9, 22, 44, 45).
The dramatic difference in kinetics for CO and NO binding has been discussed by
Ionascu et al.(44) and the temperature dependence of the kinetics was used to show that
both the Arrhenius prefactors and the enthalpic heme binding barriers are very different
for the two ligands. For example, the main rebinding channel for NO geminate
recombination is temperature independent and therefore barrierless. Other recent studies,
involving O2 rebinding to Mb, reveal two well-separated geminate phases having time-
constants of ~6ps and ~40ns, each with amplitudes of ~30%(19).
For many years it has been thought that spin selection rules are an important
underlying reason for the different dynamical behavior of the three ligands(6, 7, 10, 46,
47). The ligand fields of heme are such that the ferrous iron lies near to the spin
crossover point. Thus, a perturbation due to the presence or absence of the diatomic axial
ligand causes the spin-state to change. When the second axial ligand is not bound, the
ferrous heme iron is five coordinate and has a Hund’s rule high spin (S= 2) ground state.
When the diatomic ligand binds, the d-electrons of the ferrous iron pair to a low spin state.
108
0.01
0.1
1
10m1m10μ 100μ1μ100n10n1n100p10p
MbNO
MbO2
MbCO
N
(t)
time (s)
Figure3.1 The rebinding kinetics of diatomic ligands to ferrous myoglobin,following photolysis at room temperature in aqueous solution, are displayed ona logarithmic scale. The survival probablility, N(t), measures the population ofthe deoxyMb photoproduct as ligand rebinding proceeds. The decay times forthe three ligands span approximately 9 decades in time. In the case of CO,approximately 96% of the photodissociated CO molecules escape from theheme pocket into the solvent so that rebinding primarily involves a relativelyslow bimolecular process. In contrast, 99% of the photolyzed NO moleculesrebind geminately in two separable phases with time constants of 15ps and 170ps. In the case of O2 there are again two geminate phases, but the second phase(~50ns) is well separated from the first (~6ps). The bimolecular yield foroxygen is in the range 40-50%.
109
The spin states of ligated myoglobin are: MbCO (S=0), MbNO (S= ½), and MbO2 (S=0),
where the MbO2 complex is thought to involve Fe3+O2− antiferromagnetic coupling(48) or
triplet state spin paring in a three center π-bond(49). In the recombination process from
the initial unbound state to the final bound state, several system spin states can potentially
participate. Using angular momentum addition, we see that Mb-CO presents only a single
initial (S= 2) and final (S=0) spin state. However, for ferrous Mb-NO, the initial total
spin angular momentum could be either 5/2 or 3/2, while the final state is S =1/2. In the
case of Mb-O2, the possible initial spin states include S=3, S=2 and S=1, while the spin
state is S =0 in the final ligated form.
In theoretical considerations by Harvey(6, 50) and Franzen(7), an imidazole (Im)
ligated iron-porphine (FeP) system was used as a model, and the iron spin-state energy
surfaces for the binding of different diatomic ligands (Im-FeP-XO where X=C, N, O)
were computed. Franzen(7) used density functional theory (DFT), to calculate the
optimized ground state potential energy surfaces for each of the possible spin states as
diatomic ligand approached at three different proximal heme doming distances (0.0Å,
0.2Å and 0.4Å, respectively). The fastest geminate recombination rate was assigned to
the transition from the lowest initial spin state to the final correlated spin state (eg. Si
=1→Sf =0 for MbO2 and Si =3/2→Sf =1/2 for MbNO). The slower recombination rates
were explicitly linked to the higher initial spin states as they transformed sequentially
into the final spin states (eg. MbO2: 2→1→0 and MbNO: 5/2→3/2→1/2). However,
recent studies of MbNO recombination as a function of temperature and distal pocket
mutation(44)have definitively shown that the slower (~200ps) geminate phase for NO
110
binding is due to docking in the distal pocket, rather than to a spin selection channel
inherent to the NO ligand.
Strickland and Harvey(6) have calibrated their DFT calculations at the level of
CCSD(T) (i.e., coupled cluster single and double substitutions and triple excitations).
They have also compared both pure and hybrid DFT functionals in their calculations and
focused on the binding of CO and H2O to ferrous heme. Their NO binding calculations(6)
suffer from the appearance of a local minimum in the quartet state (S=3/2) that yields a
barrier of 1.6kcal/mol that must be surmounted in order to reach the final NO bound state
doublet (S=1/2) surface. This barrier is roughly half that calculated for MbCO rebinding
using the same methodology(6, 50). Such a barrier for NO binding to heme is
inconsistent with the temperature-independent rates experimentally observed for MbNO
and PPIXNO rebinding. Thus, at this stage, it is probably premature to use the calculated
transition-state Fe-NO bond-lengths as evidence against(6) the “harpoon”(44) and the
“product-like”(51) transition state models for NO binding that have been put forward
previously.
For CO binding, the direct quintet-singlet transition is spin forbidden so a
superexchange process, or sequential rebinding involving an intermediate S=1 triplet
spin-state, is usually invoked. Several groups(6, 7, 47) have suggested that the
superexchange (double spin flip) process is the likely CO binding channel and that this
accounts for the slower CO rebinding reaction observed in Mb(32, 42, 43, 52) compared
to the other diatoms. This possibility has also been explicitly discussed by Hopfield et al.
using a spin-orbit coupling model, operating at second order, that couples the S=0 and
S=2 states through the superexchange mechanism involving the S=1 state(10, 15).
111
These theoretical studies suggested that strong external magnetic fields (~10 Tesla)
might compete with the spin-orbit coupling to mix the iron spin-states enough to
influence the CO recombination process, particularly at low temperatures(10). The
external magnetic field influences the quantization axis for the iron spin. Spin-orbit
interactions and the internal ligand fields at the iron are dominant, but because of the
competition for the spin quantization axis, the magnitude of the (spin-orbit) coupling
matrix elements between the different spin configurations can be altered as the
eigenstates are mixed by the magnetic field. As a result, the transitions between spin-
states can be affected, leading to a potentially observable change in the ligand binding
rate.
The theoretical considerations(6, 7, 10) suggest that the diatomic heme ligands
(CO, NO, and O2) will have differences in their ligand binding and spin transition rates
that should be revealed in the Arrhenius rate expression
(1)
through differences in the prefactor, k0. In Eq. 1, kBA represents the overall temperature
dependent rate of ligand rebinding from the initially photolyzed state, “B”, to the ligand
bound state, “A”, where HBA is the enthalpic barrier for this process and kB is the
Boltzman constant. Thus, comparative studies of the magnetic field dependence of the
rebinding kinetics of these three ligands have the potential to reveal spin-dependent
effects on the rebinding rate. When such studies are carried out with extremely good
signal-to-noise, there is the possibility to reveal even relatively small effects. Such
112
studies can yield new insights into the underlying fundamental mechanisms associated
with the important life process of diatomic ligand binding in heme proteins.
The experiments presented here document our measurements of heme relaxation
and diatomic ligand recombination kinetics under high magnetic fields. We are aware of
only one other experimental study of this nature(15), which monitored a weak magnetic
field induced heme polarization anisotropy on much longer time scales and at very low
temperatures. Recent improvements in the signal-to-noise of pump-probe experiments
that are used to detect coherent oscillations in heme proteins(53, 54) offer the possibility
to observe relatively small magnetic field dependent kinetic effects. Although small
systematic changes in the amplitude of the short time (<10ps) optical response due to
vibrational relaxation were observed in some cases, no magnetic field dependent
differences in the ligand binding rates were observed beyond the noise level of the system.
Based on the absence of magnetic field dependent ligand binding rates, and on the
measurement of a wide range of Arrhenius prefactors for CO binding in several heme
systems, we suggest that either the energy gaps between the pure spin states are larger
than previously thought or that a significant mixing of the these states is already taking
place in the absence of the magnetic field.
3.3 Experimental Results
The NO recombination kinetics to ferric horseradish peroxidase (HRP) at a series
of magnetic fields are shown in Figure 3.2(a). A maximum entropy method (MEM) was
used to fit the data(55) and the rate distributions that fit the observed kinetics are
presented as an insert in the figure. It can be seen that the rate distributions obtained at
113
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
(b) Ferrous MbNO
MEMfit 0 Tesla 10 Tesla
Δ A
(arb
itrar
y un
its)
Time (ps)
0.0
0.2
0.4
0.6
0.8
1.0 (a) Ferric HRP-NO
MEMfit 0 Tesla 2 Tesla 4 Tesla 6 Tesla 8 Tesla 10 Tesla
Δ A
(arb
itrar
y un
its)
106 108 1010 10120.0
0.4
0.8
1.2
Rate (s-1)
106 108 1010 10120.0
0.2
0.4
0.6
Rate (s-1)
Figure 3.2 (a) Recombination kinetics of ferric HRP-NO as a function ofmagnetic field. The sample was stabilized for 30 minutes after each fieldintensity change. The pump and probe wavelengths were 403nm and 420nm,respectively. Black solid lines are the MEM fits and the insert shows the MEMrate distributions as a function of magnetic field.(b) Comparison of the ferrous MbNO rebinding kinetics with (H=10 T) andwithout application of a magnetic field. No difference outside of experimentalerror is observed. The inset shows the MEM rate distributions.
114
the different field strengths agree quite well. Figure 3.2(b) shows a similar experiment
conducted on ferrous MbNO. The invariance of both the raw kinetic data and the MEM
analysis points to the absence of a measureable magnetic field effect on the NO binding
reaction in ferric HRP and ferrous Mb.
Figure 3.3 shows the geminate kinetics and the magnetic field response of the NO
complex of (Fe2+)PPIX under two solvent conditions (80% glycerol and 1% CTAB).
The data again clearly demonstrate that the applied magnetic field leads to no measurable
effect on the heme-NO rebinding.
Figures3.4 and 3.5 present the effects of strong magnetic fields on the binding of
CO to FePPIX. The CO complex of FePPIX in the absence of 2MeIm was chosen for
study because the CO binding is much faster when water (or OH-) is in place as the heme
proximal ligand(56). The high repetition rate lasers used in these studies generate
excellent signal to noise, but do not allow the study of the slower rebinding systems such
as MbCO and 2MeIm-FePPIX-CO because these samples do not fully reset to
equilibrium within the arrival time (5.3μs) of the pump-probe pulse pairs. The FePPIX-
CO sample is studied in 80% glycerol because the spin and optical properties of FePPIX
and FePPIX-CO under this condition mimic those of deoxy Mb with surprising accuracy,
especially when 2MeIm is bound as the axial ligand. In the absence of 2MeIm, the optical
transitions are blue shifted by ~10nm, but, as can be seen in the insert to Figure3.6, there
is a clear isosbestic point as the CO rebinds under these conditions(56).
On the insert of Figure3.5, statistical error analysis using sets of 10 runs, each
with 99 logarithmic sample points, reveals the entire kinetics in roughly 30 minutes. The
115
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0 (b) Fe(II)PPIX-NOin 80% glycerol
MEMfit 0 Tesla 14 Tesla
Δ A
(arb
itrar
y un
its)
Time (ps)
0.0
0.2
0.4
0.6
0.8
1.0 (a) Fe(II)PPIX-NO in Kpi with 1% CTAB
MEMfit 0 Tesla 10 Tesla
Δ
A (a
rbitr
ary
units
)
109 1010 1011 10120
1
2
3
Rate (s-1)
109 1010 1011 10120
2
4
Rate (s-1)
Figure3.3 Comparison of NO rebinding kinetics to Fe2+-protoporphyrin IX withand without an applied magnetic field. The pump is 403nm and the probe is435nm nm. The upper panel (a) is for a CTAB solution and the lower panel (b)is for a glycerol solution. No difference outside of experimental error isobserved.
116
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
Fe(II)PPIX-COin 80% glycerol
MEMfit 0 Tesla 10 Tesla
Δ A
(arb
itrar
y un
its)
Time (ps)
109 1010 1011 10120.0
0.5
1.0
1.5
2.0
Rate (s-1)
Figure 3.4 Comparison of the CO rebinding kinetics to Fe2+-protoporphyrin IXin 80% glycerol with and without the application of a magnetic field. Thesample is pumped at 403nm and probed at 435nm. The black solid lines are theMEM fits of data and the rate distributions are displayed in the inserts. Themaximum in the data trace is arbitrarily normalized to unity and represents thecut-off time beyond which data are analyzed. The time zero is taken at the halfheight of the rising signal.
117
Figure3.5 Detailed measurements of the magnetic field dependence ofFePPIX-CO in 80% glycerol pumped at 403nm and probed at 435nm. Themeasured rate distributions are the same as shown in Fig. 7. The amplitude ofthe 5ps process appears to vary systematically with applied magnetic field.The insert shows two of the kinetic traces renormalized to one at time zerousing an exponential function to fit the ~5ps component. This allows a bettervisualization of that the amplitude of the fast response is changing rather thanthe rate constant(s). The error bars for the measurement are barely discernablein the figure.
10-1 100 101 102 103 1040.0
0.5
1.0
N (t
)
0 Tesla MEMfit10 Tesla MEMfit
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
Fe(II)PPIX-CO in 80% glycerol
0 Tesla -1st 10 Tesla -2nd 7 Tesla -3rd 4 Tesla -4th
1 Tesla -5th
0 Tesla -6th
Time (ps)
Δ A
(arb
itrar
y un
its)
118
standard deviation of each data point along the N(t) axis is on the order of ~1%, while the
laser jitter along the time axis is less than 800fs. As shown, the application of strong
magnetic fields to heme protein samples leads to no detectable effect on the kinetic rates.
The peak of the kinetic data near 3ps represents the maximum value of the signal and this
is the time point after which the kinetics are fit in order to generate the MEM rate
distributions. By comparing kinetics, where the underlying rate is systematically varied
we find that the experimental data can be used to set a limit on magnetic field-induced
changes of the observed geminate kinetic rate constant, kg. After including the possibility
of systematic errors due to optical geometry changes and fitting procedures, this limit is
found to be Δkg/kg ~ ±5x10-2.
As shown in Figure3.4, following photolysis of FePPIX-CO, a magnetic field
effect can be discerned in the amplitude of the early time (<10ps) optical response.
Figure3.5 explores this effect in more detail and indicates that it is systematic. The insert
in Figure3.5 shows the zero field and 10T kinetics re-normalized by fitting the “fast”
~6ps phase of the response with an exponential function and then extrapolating back to
time. The magnetic field induced change in the overall kinetics is well described by a
simple change in this “fast” phase amplitude from roughly ~55% at 0T to ~45% at 10T.
SRC model(11, 57) plus an exponential decay were used to fit the kinetic trace of
PPIX-CO in Figure3.5 under 0T field and 10 Tesla respectively.
∆√
√∞ √ (2)
The fitting parameters are compared in Table 3.1. Corresponding fittings with
experimental data are displayed on figure 3.6.
119
A C t0 (ps) P1 P2 τ(ps) P0
0 Tesla 0.93 1.85 5.6 0.73 0.72 5.5 0.27
10 Tesla 0.95 1.82 5.4 0.70 0.54 5.5 0.29
Table 3.1 Fitting parameters of PPIX-CO in 80% glycerol/water mixture under 0Tesla and 10 Tesla magnetic field
120
We want to stress at this point that the “fast” optical response, observed for the
FePPIX-CO sample in Figures 3.4 and 3.5, certainly involves a rapid (~0.3-5ps) spectral
diffusion (cooling) response of the hot deoxy FePPIX photoproduct(37), but it may also
include an underlying fast component of CO rebinding, which has been previously
observed at low temperature and is sometimes referred to as process I*(58, 59). We note
that the observed signal at 435nm between 1-10ps also involves a convolution of the
instrument function.
Because of the potential for interference between the rebinding and cooling
signals, we decided to examine the kinetics at other wavelengths. Figure 3.7 shows a
comparison of the 0T data in Figure3.4 probed at 435nm (blue stars) to that (red dots)
obtained at 410nm (i.e., the peak of the transient bleaching signal) using another pump-
probe instrument with much better (~100fs) time resolution(56). We also include another
0T kinetic trace taken at 424nm (i.e., the peak of the transient anti-bleaching signal) using
the fs/ps magnet-based system (orange triangles).
Independent examination of the spectral response of the photoexcited deoxy heme
in glycerol solution reveals a bleaching signal near 424nm (Figure3.8). This observation
helps to explain why the transient absorption signals observed at 424nm for t<10ps are so
much weaker than those observed at 435nm. The CO rebinding signals at 424nm on this
timescale have an opposite sign (i.e., increased transient absorption) compared to the
deoxy heme transient cooling signal (decreased transient absorption). Thus, at 424nm,
any fast (<10ps) CO rebinding signal would tend to be cancelled by the oppositely signed
cooling signal of deoxy heme whereas at 435nm the cooling and rebinding signals are
additive.
121
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
Kinetics Simulationfor PPIX-CO in 80% glycerol/water mixture
0 Tesla Fitting for 0 Tesla 10 Tesla Fitting for 10 Tesla
Δ A
Time (ps)
Figure 3.6 Kinetic traces of PPIXCO (pumped at 403 nm and probed at 435nm) in different values of the magnetic field in 0 Tesla and 10 Tesla. Thesolid lines represent the fits using equation (2).
122
380 390 400 410 420 430 440 450
-0.04
0.00
0.04
ΔA (O
.D.)
nm
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Time (ps)
424nm
435nm
410nm
Δ
A (a
rbitr
ary
units
)
PPIX-CO in 80% glycerol
Figure 3.7 The CO binding kinetics of FePPIX (pumped at 403nm and probedat 435nm) taken with the fs/ps pump-probe laser system (blue stars) arecompared with the kinetics taken with a 100fs time resolved system (fs/fs)probed at 410nm (-ΔA410 is plotted as red dots). Additional data taken with thefs/ps system pumped at 403nm and probed at 424nm are shown as the orangetriangles. The data are scaled to equal values at long times in order to revealthe short time deviations arising from the instrument function when itconvolves with a fast (0-5ps) thermal response and/or rebinding signal. Theinsert shows the spectral evolution of this sample at room temperature whenprobed using 100fs continuum pulses. The small solid arrows show the probewavelengths for the data in the main figure.
123
0.1 1 10 100 1000 10000-500
0
500
1000
1500
2000
2500
3000
3500
Responses of PPIX-CO and deoxy PPIXPump at 403nmProbe at 424nm
PPIX-CO deoxy-PPIX
Δ A
Time (ps)
Figure3.8 Spectral responses of PPIX-CO and Deoxy PPIX in 80%glycerol/water solution. The photoexcited deoxy heme reveals a bleachingsignal near 424nm. The Y-axis indicates the lock-in amplifier readings underthe same pump-probe conditions for the CO bound and deoxy PPIX samples.
124
In an attempt to remove some of the ambiguity introduced by heme cooling
signals and the fs/ps instrument response, we also carried out room temperature
experiments using the FeCO infrared transition at 1954 cm-1 (Figure3.9). It is clear from
these experiments that approximately 10% of the CO photolyzed population rebinds in
the first 10ps. However, the analysis of the t < 10ps IR data is consistent with a
straightforward extension of the distributed coupling model that has been previously
applied 11 to fit the kinetics for t>10ps, so it does not appear that a separate ultrafast
exponential CO rebinding phase (attributable to the I* process) is present. This, along
with fits to the data at 435nm shown in Figure3.6 indicates that the fast exponential phase
(time constant ~5-6ps) is due to cooling of the deoxy heme photoproduct.
Figure 3.10 displays measurements of geminate recombination for the oxygenated
L29W Mb mutant (L29WMbO2). This mutant is used because of its enhanced geminate
rebinding amplitude, which is sufficient to allow reset to equilibrium between the pump-
probe pulse pairs. There is a barely perceptible difference in the data trace when the 10T
magnetic field is applied, but the MEM derived kinetic rate distributions shown in the
insert do not show a significant variation. Thus, the small changes perceived in the O2
kinetic data do not arise from rate changes, but rather from a possible small change in the
geminate amplitude. The bottom panel of the figure shows the data renormalized to unity
by use of an exponential fit to the data that is extrapolated to time zero. Here the
potential for a small change in the geminate amplitude is more easily visualized.
125
1 10 1000.0
0.2
0.4
0.6
0.8
1.0PPIX-CO in 80% glycerol
IR transient data, 1954cm-1
MEM analysis
Δ
A
Time (ps)
108 109 1010 1011 1012
0.0
0.1
0.2
0.3
0.4
0.5
Figure3.9 Pump-probe kinetics measurement at the ν(C-O) stretch modewavelength, 1954cm-1 . The blue solid line is the MEM fitting of theexperiment data. Corresponding rate distribution is displayed on the insert.
126
Figure 3.10 Comparison of L29WMbO2 rebinding kinetics with (H=10 T) andwithout application of a magnetic field pumped at 403nm and probed at 435nm.The MEM rate distribution shown in the insert reveals an exponential geminateprocess that is independent of the magnetic field. The upper panel shows theMEM fits when the data are normalized to one at the maximum data point,which is the cut-off time beyond which data are analyzed. The time zero istaken at the half height of the rising signal. The lower panel shows the data andfits extrapolated back to unity at time zero by using an exponential fittingfunction.
1 10 100 1000 10000
0.2
0.4
0.6
0.8
1.0 MEMfit 0 Tesla 10 Tesla
N (t
)
Time (ps)
0.2
0.4
0.6
0.8
1.0L29WMbO2
Δ A
(arb
itrar
y un
its)
106 108 1010 1012
0
2
4
6
Rate (s-1)
127
3.4 Discussion
Under the assumption that the spin of the iron-ligand system plays an important
role in determining the reaction rate, we can introduce a very simple model to estimate
the perturbative effect of the applied magnetic field on the Arrhenius prefactor (k0 in Eq.
1). The prefactor incorporates all of the non-enthalpic factors (such as entropic barriers,
attempt frequencies, frictional effects, and spin tunneling matrix elements) that affect the
reaction rate in addition to the enthalpic barrier. Here we use a simple three level system
that involves a (ligand bound) ground state as well as two “excited” unbound states, one
with “allowed” (a), and one with “forbidden” (f ), propensity for making the spin
transition to the ligand-bound ground state. In addition to spin-orbit coupling the
magnetic field can perturb and alter the mixing of these states so that the amount of
allowed transition amplitude from state a mixed into state f can be altered by the
magnetic perturbation. Quantum admixtures of spin states differing by ΔS=1 have been
treated previously by Maltempo(60).
As a specific example, we can imagine the ground state to be the CO bound
singlet and the “allowed” and “forbidden” states to be the deoxy triplet and quintet states,
respectively. We take the allowed triplet state to be of higher energy as CO approaches
the deoxy heme. The energy gap, Ea-Ef, between the triplet and quintet states of the
equilibrium deoxy heme, denoted as ΔEaf, is thought to be relatively small(6, 7, 61). Both
“excited” states (a and f) are assumed to be far removed (i.e., by the CO binding energy)
from the 6th ligand bound ground state when the nuclei are in equilibrium. The
perturbation of the spin-dependent Arrhenius prefactor can be calculated as a quantum
mechanical transition rate, where the applied magnetic field mixes the allowed and
128
forbidden states. We take δB to be a measure of the magnetic field induced mixing that
couples the allowed (a) and forbidden (f) states. The magnitude of the perturbative
mixing term, δB/ΔEaf, can then be estimated based on the experimental detection limits.
To do this, we take CO binding in Mb to be an experimental limiting case of a “spin-
forbidden” prefactor (k0f ~109s-1) and NO binding as a limiting case for a “spin-allowed”
prefactor (k0a~1011s-1). Application of simple perturbation theory within the subspace of
the allowed and forbidden states then leads to mixing of the states and an approximate
expression for the square of the perturbed probability amplitude (i.e., the perturbed
prefactor, k0*). Taking Δk0f=k0*-k0f as the difference between the perturbed and
unperturbed prefactors for the “forbidden” CO binding reaction, we find
∆ 2
∆
/cos ∆
∆ (2)
The leading term in the magnetic field perturbation carries a phase factor, cos ∆ ,
because it involves the interference term in the Golden Rule expression for the decay rate
of the magnetically perturbed initial state |∆
| as it evolves into the ligand
bound singlet ground state. The phases of the complex matrix elements involved in
coupling the ground state with the “allowed” and “forbidden” states are unknown and ∆
is simply the difference of these two phases. Taking 5x10-2 as the detection limit for
fractional change in the observed rate, and a maximum value for the phase factor
(cos ∆ 1) leads to ∆
2.5 10 . A similar calculation for the minimum
phase (cos ∆ 0), using the second order term, leads to ∆
2.2 10 . Thus, a
typical magnetic field induced mixing perturbation of δB~10cm-1, leads immediately to
129
ΔEaf > ̃ 500cm-1 for the minimum phase, while ΔEaf > ̃ 4000cm-1 if the phase factors are
such that they maximize the prefactor change upon application of a magnetic field. Thus,
insofar as spin selection rules between “pure” spin states play a role in these reactions,
the experiments reported here can be used to set approximate limits on the average
energy separation between the forbidden and allowed spin levels as the transition state is
approached along the Fe-Ligand and heme doming coordinate(11, 57). Prior calculations
indicate a low lying spin S=1 triplet state within 300cm-1 of the ground state in order to
account for the Mossbauer spectra of the deoxy heme system(62). More recent DFT
calculations actually suggest that the triplet state energy can sometimes fall below the
quintet state(6). Thus, the size of the energy gap between the triplet and quintet states
that is needed to eliminate detectable magnetic field effects is surprisingly large.
On the other hand, it remains possible that spin admixtures are present at room
temperature, even in the absence of the applied magnetic field. The presence of such
admixtures is consistent with early far infrared magnetic resonance measurements on
high-spin ferrous heme in Mb and Hb, where a simple S=2 spin Hamiltonian was found
to be insufficient to account for the data(61). Magnetic susceptibility measurements have
been interpreted to indicate that a “pure” S=2 spin is the likely 290K ground state for
ferrous heme in Mb and Hb. However, uncertainty in the magnitude of the orbital
component that generates the observed µeff =5.5µB(12-14) makes unequivocal
assignment of a pure S=2 ground state difficult.
In the process of this investigation we studied samples with a variety of possible
spin channels (Table 3.2). For example, the binding of NO to ferrous heme involves two
potential spin transitions; one is an “allowed” first order transition (ΔS =1 with an “initial”
130
Sample Ligand SL SiTot Sf
Tot ΔS k0(H) k0 (s-1) Ref
PPIX (Fe2+,S=2)in glycerol
CO 0 2 0 2 no 1.5x1011 11
L29WMb (Fe2+, S=2) O2 1 3 0 3 no 1.8x1011 (Unpublished)2 0 21 0 1
Mb (Fe2+, S=2) NO 1/2 5/2 1/2 2 no 9.0x1010 433/2 1/2 1
PPIXa (Fe2+, S=2) NO 1/2 5/2 1/2 2 no 1.2x1011 433/2 1/2 1
HRP (Fe3+, S=5/2) NO 1/2 3 0 3 no >6.0x1010 162 0 21 0 1
Table 3.2 Spin changes associated with ligand binding to high-spin heme iron
a In equilibrium in 80% glycerol S=2, however when NO binds, L1 dissociates, so that the geminate rebinding may involve a transient S=1 state.
131
spin, Si=3/2, and a “final” spin, Sf=1/2) and one is a “forbidden” transition (ΔS=2, with
Si=5/2 and Sf=1/2) that can proceed via either a “sequential” or a second order
“superexchange” process. In fact, it has been suggested(7) that the “fast” (~10ps) and
“slow” (~200ps) geminate recombination phases observed(33, 44, 45) for MbNO binding
are due to the allowed and forbidden (sequential) spin channels, respectively.
As direct evidence to the contrary, we note that there is a single ~10ps
temperature independent(44) exponential geminate phase observed in Mb mutants that
have the xenon pocket blocked. This demonstrates that spin selection is not responsible
for the slower geminate rebinding phase of MbNO. Moreover, the temperature
dependent measurement of the rates in native MbNO demonstrates that the prefactor for
both the “slow” and the “fast” phase is ~1011 s-1(44). Thus, as suggested previously, we
believe that the slower geminate phase results from a small enthalpic barrier involving
the return of the NO ligand from a distal docking site near the heme, probably involving
the Xe4 pocket(44). The model invoking a distal pocket docking site also explains why
only the relative amplitudes, but not the rates, of MbNO binding are affected by external
perturbations such as glycerol containing solvents(45), pumping wavelengths(44), or
mutations(44).
An analogous situation is documented in Figures3.4 and 3.5, where it can be seen
that the application of the magnetic field affects the amplitude, but not the rates, of the
fast optical response for PPIXCO binding. However, we must recognize that the sub-
10ps optical response at 435nm contains a significant contribution from the cooling of the
transient deoxy heme state as well as from any underlying CO rebinding signal that might
be present. Our fits to the room temperature PPIXCO rebinding kinetics, using the IR
132
data to eliminate the heme cooling signals, demonstrates that a separate sub-10ps
exponential CO rebinding phase is not present. Thus, the exponential response observed
at 435nm is assigned to transient cooling of the deoxy heme photoproduct.
When we fit the data in Figure3.4, using a superposition of the SRC rebinding
model(11, 57) along with a separate exponential phase to account for the short time heme
cooling, we find the exponential component to have a time constant of 5.5ps and an
amplitude that decreases by ~10% in going from 0 and 10T (see Figure 3.6 and Table
3.1). Using the same fitting protocols, we find that the exponential phase has a vanishing
amplitude when applied to a data set that is composed of a superposition of the IR
response t<40ps and the optical response t>40ps. From this we conclude that the
amplitude of the 5.5ps exponential heme cooling response changes as a function of
applied magnetic field. Independent studies of the deoxy heme cooling using improved
100fs time resolution reveal that the cooling response at 435nm has a time constant of
~4ps (unpublished), which is in good agreement with the present results, given that the
instrument response of the fs/ps laser system increases the time constants in this range by
~20%.
One possibility that would account for the observation of a magnetic field
dependent cooling amplitude involves a hypothesis where the amount of vibrationally hot
electronic ground state heme is slightly reduced as the applied magnetic field is increased.
This would lead to a smaller deoxy heme cooling signal and it would therefore be
consistent with the observed reduction in the sub-10ps exponential amplitude at higher
magnetic field strength. This is an unexpected result, and additional studies are necessary
in order to further evaluate this possibility. If magnetic field effects alter the ultrafast
133
non-radiative decay pathways associated with CO photolysis and the deoxy heme cooling
process, one might generally expect that both the rates and amplitudes might be affected.
However, the branching ratio for a prompt non-radiative transition into the vibrationally
hot electronic ground state (following CO photolysis) could be less than unity(9). If this
is the case, and/or the branching ratio is further reduced by the applied magnetic field, the
observed vibrational cooling rates in the ground electronic state should remain fixed but
the amplitude of this signal would be decreased. Another obvious hypothesis involves a
magnetic field dependent reduction in the CO quantum yield and the cooling of a residual
hot six-coordinate CO bound species. However, this hypothesis predicts an increase,
rather than a decrease, in the amplitude of the sub-10ps response and we therefore
exclude this possibility.
A very weak magnetic field dependence of the O2 binding reaction in Mb (L29W)
may also be present. Figure3.10 shows that, although there are indications of a very
small magnetic field induced change in the geminate amplitude, the MEM analysis
reveals no statistically significant change in the rebinding rate distribution. In contrast to
NO binding, the preliminary temperature dependent kinetic measurements of the O2
binding reaction in wild type Mb (See Chapter4) indicate that the prefactors for the two
geminate phases seen in Fig. 3.1 are quite different(19). The ~5ps geminate phase has a
prefactor near 1011s-1, while the ~40ns geminate phase has a prefactor near 109s-1. This
could indicate the presence of either a spin selection mechanism, or an entropy
production timescale(11) for the photoproduct that falls in the range between 10ps and
10ns and leads to a larger entropic barrier for the slower geminate phase.
134
Finally, we display in Table 3.3(63-67) the geminate rebinding rates and
Arrhenius prefactors for CO binding to several different heme systems. The significant
differences observed for the CO geminate rate demonstrates that there is not a universal
spin selection rule for CO binding that acts as a consistent limiting factor in these
reactions. The net spin change upon ligand binding in these systems is the same for each
of the equilibrium species (ΔS=2), yet the rates are very different. We do not believe that
this variation is due to non-equilibrium photoproduct species involving triplet states
because the transient absorption spectra (e.g., see insert to Figure3.7) indicate that a
genuine ΔS=2 transition is taking place over a wide range of timescales for the different
samples. For example, a detailed analysis(11) of the temperature dependence of CO
binding to the FePPIX model complex reveals a prefactor for CO binding near 1011s-1,
which is similar to the “spin-allowed” prefactor for NO binding. Other heme systems,
such as microperoxidase(63) , carboxymethylated cytochrome c(67), mutants of
cytochrome c(68) , and CooA(66, 69) all have CO geminate rebinding rates that are
faster than 1010s-1 (see Table 3.3), which sets a lower limit for their Arrhenius prefactors.
Additional temperature dependent studies are needed to establish the precise
magnitude of these prefactors, but it is clear from the rapid rebinding kinetics of various
heme systems, that CO binding is not generally being retarded by an unfavorable (ΔS=2)
spin selection rule. In fact, it appears that the unusually small prefactor (~109s-1) found
for CO rebinding to Mb may be anomalous, rather than the general rule for heme-CO
rebinding. The possibility that these “fast” CO binding reactions take place through
genuine population of a S=1 transition state (i.e., via a sequential rather than a
135
Protein kg (109 s-1) Ig kBAa (109 s-1) k0 (109 s-1) reference
WT Mbb 0.006 4% 0.0002 1 29
Mb (V68W)b 0.006 76% 0.005 0.88 Unpublished
PPIX+2MeIm(95%Gly)c 0.83 20% 0.17 > 0.17 55
NP4 (pH=5.0)c 5.1 99% 5.1 >20 Unpublished
HRP+BHAb 2 90% 1.8 > 1.8 16,63
PPIX (95% Gly)c 20 95% 19 150 55
MP-11 (aggregated)c 16 78% 13 > 13 62,64
CooAd 13 60% 8 > 8 65
Cm Cyt Ce 63 27% 17 > 17 66
Table3.3 Kinetics of CO binding to selected heme proteins
a To extract the kBA from these data we used a simple three state model55.Fitting methods for CO geminate recombination : bSingle exponential fit.cStretched exponential fit. dTwo exponential fit, only the fast component ispresented here and used to estimate the value of the kBA. eThree exponentialfit, only the fast component is presented here and used to estimate the valueof the kBA
136
superexchange mechanism) or that entropy production timescales and/or ligand
confinement are involved in determining the prefactor will be the topic of future work.
137
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143
Appendix 1 Oxygen Rebinding Kinetics to Myoglobin and its Mutants (V68W, L29W)
with Temperature Variation in Aqueous Solutions and Glycerol Mixtures
A1.1 Experimental Methods
Sample Preparation. Detailed experimental methods are explained and discussed within
Chapter 2. Here, in connection with the oxymyoglobin (and its mutants) preparation, two
methods are applied here. Method 1 below corresponds to the first method of preparation
for MbO2 described in Chapter 2 and method 2 below corresponds to the third method of
preparation described in Chapter 2. The second method described in Chapter 2 can not be
used for low temperature studies because the glycerol solution does not pass through the
G25 column.
Method 1: Ascorbic acid with air. Stock solution of metmyoglobin is diluted within a
cylindrical glass vial. The concentration of the diluted sample is tested with a 1mm quartz
cuvette. The final concentration is adjusted to approximate 1 O.D. at its Soret peak
(409nm). Then, a tiny amount of ascorbic acid powder is added into the metmyoglobin
solution. The glass vial sits in the open air stationarily for about 2 hours (for metMb) or
less (for V68W Mb) until a clear dividing line within two layers of species appears. The
top layer of substance is transported into the spinning cell and ready for the experiment.
144
300 400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
580nm542nm
415nm
MbO2prepared by method 1
Before Exp. After Exp. O
.D.
Wavelength (nm)
Figure A1.1 Absorption spectrum of an MbO2 sample prepared with method1 and its absorption spectrum comparison before and after a series oftemperature dependent kinetics experiment.
145
Figure A1.1 shows absorption spectrum of an MbO2 sample prepared with this method
and its usual spectral change after a series of temperature dependent kinetics experiment.
Method 2: Sodium dithionite with oxygen gas. Diluted metMb sample solution within the
glass vial is sealed with a septum cap and parafilm. Then it is vacuumed and Argon
flushed as in the standard degassing procedure. Meanwhile, assemble and seal the
spinning cell, use the vacuum pump to pull out the air inside the cell. After that, transport
3 ml metMb solution into the vacuumed cell with a needle syringe piercing through the
rubber sleeve stopper septum for it. Then, adding appropriate amount of sodium
dithionite solution into the cell, the sample’s Soret peak moves to 435nm. Adjust the
output pressure of the oxygen cylinder to the minimum value. The oxygen bubbles going
through a bottle of buffer should be observed as single, inconsecutive ones, not a bubble
line as in the CO flushing. Introduce this pure oxygen gas into the sealed cell promptly
for about 1-2 seconds. First, it will use up all the extra sodium dithionite molecules and
then, oxygen molecules (O2) bind deoxymyoglobin to form oxymyoglobin. Dramatic
color change of the sample solution could be observed during this oxygen association
process. After ensuring that all the deoxymyoglobin is transformed to oxymyogobin, the
last step is to use a small-bore needle, piercing through the sleeve stopper cap for less
than 1 second, bring down the gas pressure inside the cell to 1 atm.
Figure A1.2 shows absorption spectrum of an MbO2 sample prepared with
method 2 whose Soret peak locates at 418nm instead of 415nm which appears in the
preparation with method 1. Figure A1.3 compares the absorption spectra of horse heart
oxymyoglobin prepared with method 1 vs. method 2.
146
300 400 500 600 7000
1
580nm543nm
418nmMbO2prepared by method 2
O
.D.
Wavelength (nm)
Figure A1.2 Absorption spectrum of an MbO2 sample prepared with method 2
147
Figure A1.3 Absorption spectra comparison of horse heart MbO2 sampleprepared with method 1 vs. method 2. (the absorption data were normalized to1 at the Soret peak for illustration purposes.)
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
543nm, 580nm
418nm415nm
Method 1 method 2
Comparison of MbO2prepared with different method
O
.D. (
arbi
trary
uni
ts)
Wavelength (nm)
148
Experience and practice are needed to observe the color changing to control the oxygen
flushing. However, even with over-flushing of the oxygen to the sample, which is
registered as a double peaks at the α/β band (542nm &580nm) but the Soret band peaks
around 409 or 410nm (Figure A1.4), it’s not the end of the world. Keep gas-flushing and
oxidize all the samples back to the met form until the double peak feature at the α/β band
is disappeared, then repeat the preparation procedure described above. In several tests, no
difference is observed for this refurbished sample and a freshly made sample in
absorption spectra and pump-probe kinetics. It is worth to know this, especially while
treating the precious V68W mutant. Figure A1.5 shows the absorption spectrum of a
V68W MbO2 sample prepared with method 2 and its usual spectral change after a series
of temperature dependent kinetics experiment.
Myoglobin L29W was provided by Professor John Olson at Rice University in its
ferrous, O2 bound form. A1.6 shows the absorption spectrum of a diluted L29W MbO2
sample and its usual spectral change after a series of temperature dependent kinetics
experiment.
It’s very important to monitor the sample absorption spectral evolvement before
and after the experiment. MbO2 samples are very easy to be oxidized and change to a
mixture of different species. Abnormal kinetics traces could be obtained once this happen.
A lot of factors could trigger this sample evolvement. Extreme care needs to be taken
here.
149
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Mixture Sample of MetMb/MbO2
410nm
580nm542nm
O
.D.
Wavelength (nm)
Figure A1.4 Absorption spectrum of an MbO2 prepared by method 2, butover-flushed with uncertain amount of excess oxygen gas.
150
300 400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
V68W MbO2prepared by method 2
Before Exp. After Exp.
O
.D.
wavelength (nm)
Figure A1.5 Absorption spectrum of an V68W MbO2 mutant sampleprepared with method 2 and its absorption spectrum comparison before andafter a series of temperature dependent kinetics experiment.
151
300 400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
before Exp. after Exp.
L29W MbO2
O.D
.
Wavelength (nm)
Figure A1.6 Absorption spectrum of an L29W MbO2 mutant sample and itsabsorption spectrum comparison before and after a series of temperaturedependent kinetics experiment.
152
Experimental Configuration. Temperature control and experimental setup are described
in Chapter 2. For all the ultrafast kinetics (3ps-13ns), the pump wavelength is set to
403nm and the probe wavelength is set to 435nm. The pump pulse energy is around 5nJ
and that for the probe pulse is around 0.5nJ as they arrive at the sample. For the longer
time kinetics measurement, laser flash photolysis system is used. Briefly, laser pulses (at
532nm) generated by a 10Hz Nd-doped yttrium-aluminum-garnet (YAG) laser
(Continuum, Inc.), is used to excite the sample. A cw beam produced by a universal arc
lamp (Oriel Instruments, Model 66021) is used to probe the sample evolution. After
passing through the sample, the probe light is sent into a 0.25m monochromator (Oriel
Instruments, Model 77200). Kinetic response at certain wavelength selected by the
monochromator is detected by a photomultiplier (Hamamatsu, H6780) and recorded with
a broad band digitizer (Lecroy 9420).
4.2 Results and Data Analysis
Kinetic models and prior work on MbO2. Zewail et al. (PNAS, Vol. 101, Issue 52,
Pages 18000-18005, 2004) studied the oxygen recombination to human WT myoglobin in
the same time window from picosecond to 10 nanoseconds. Basically, they investigated
this O2 rebinding kinetics at room temperature with mutation variance. Intentionally or
unintentionally, two its mutants (V68F and I107F) are chosen to compare with the wild
type human myoglobin. In all three cases, no mutation induced difference is observed on
the picosecond scale but drastic differences are on the nanosecond time scale. Their
investigation led to an explanation as “bifurcation model”. In this model, O2 rebinding to
myoglobin is divided into two categories: the directed population and the undirected
population. The directed population will bind back within the few picoseconds and be
153
little affected by the protein immediate environment around the heme binding site. The
undirected population recombination can be affected by thermal and intra-molecular
motions on a longer time scale.
Here, in my studies, another two mutants (V68W and L29W) are chosen to
compare with native horse heart myoglobin for kinetics investigation. Contrary to what
they observed, this “directed population” recombination is actually affected by the
mutation. A new kinetic model is needed to describe this important kinetics.
Figure A1.7: Temperature dependent kinetics data of MbO2 and its oxidation product
MetMb is shown. MbO2 sample is prepared from MetMb with Method 1. All the kinetics
traces are normalized at the peak values. MbO2 Kinetics at 296K is measured first and
then repeated after two other kinetics measured. A very good agreement is reached here.
Equilibrium absorption spectra are compared before and after the kinetics measurement
and found to be equivalent (Shown on figure A1.1). MbO2 displays two decaying phases
within the detectable temporal windows. The first fast phase decays single-exponentially
and ends at 20ps. No temperature dependence of its rate is obtained within the setup
detection limitation. A very weak dependence of its amplitude related to the temperature
variation is observed. The second phase, which starts around 1 nanosecond, was only
partially detected here due to the restriction of setup measuring range. Clear temperature
dependence is shown on the early stage of this slow phase. Because of the incomplete
information, no conclusion can be drawn about the amplitude dependence on temperature
based on this work. The pump-probe responses of metMb are also listed in this figure. It
is revealed unambiguously that there is no temperature dependence of this decay from the
laser-excited state, both for the rate and amplitude. Rising edges of these two types of
154
0.1 1 10 100 1000 10000
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
MEM fit for the rising edges:
MbO2 MetMb
3.2ps1.7ps
Temp-Dependenceof MetMb:
Temperature Dependence of MbO2: 296K-1st
316K-2nd 276K-3rd 296K-4th
275K 296K 318K
Δ
A
Time (ps)
-20 -15 -10 -5 0 5 10 15 200.0
0.2
0.4
0.6
0.8
1.0
Time(ps)
Δ A
Figure A1.7 Temperature dependent kinetics data of Horse Heart MbO2 andits oxidation product MetMb.
155
pump-probe responses (O2-rebinding vs. hot spices cooling) are fitted with MEM method.
These two types of responses can also be differentiated by the time at which the ΔA
maximum is reached (3.2ps vs. 1.7ps). Insert: experiment data displayed linearly for the
MetMb for -20ps -20ps. It shows that the response is approaching the instrumental
function, which is the convolution of a 200fs pump pulse and a ~3ps probe pulse.
Figure A1.8: Another temperature dependent kinetics study of MbO2 from 276K to
316K in step of 10K. The sample is made with Method 1 within borate buffer (pH=8.42).
Black solids are fittings for each individual kinetic trace. The discontinuities of the fits
are results from two different fitting methods: from ~3ps to 400ps, the data are fitted with
a single exponential decay; after 400ps, the kinetics fittings are extracted from the MEM
data fittings for that range. The single exponential fittings are back-extrapolated with the
fitting functions to time zero and normalized to make N (0) =1. Related fitting parameters
are listed on Table 4.1. Insert: MEM fittings for kinetics from 400ps to 10ns are shown in
linear-linear plot, with normalization to unit value at the starting points. It clearly shows
that as the temperature increased, the slow recombination process is getting faster.
Figure A1.9: O2 recombination kinetics to Horse Heart Myoglobin in 0.1M borate buffer
(pH=8.42) at room temperature (T≈296K). The oxymyoglobin sample for the ultrafast
pump-probe experiment (<13ns) is prepared with Method 1. The whole range data set
consists of three sets of data: for the longer time (>10ns) kinetics, flash photolysis system
which has 10 Hz repetition rate is used; from 3ps to 13ns, the data was taken by a
synchro-lock pump-probe system which has a repetition rate of 190 kHz. The data
simulation from 0ps to ~3ps is generated from back-extrapolation of the fitting of data
from 3ps to 400ps, which is 0.72 0.28 . . This kinetics trace, spanned 11
156
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
2000 4000 6000 8000 100000.75
0.80
0.85
0.90
0.95
1.00
Temperature DependentKinetics of MbO2
276K 286K 296K 306K 316K
N
(t)
Time (ps)
Time (ps)
ΔA
Figure A1.8 Temperature dependent kinetics study of MbO2 from 276K to316K in the step of 10K. Insert: MEM fittings for kinetics from 400ps to 10nswith normalization at the starting points.
157
Table A1.1 Temperature Dependence of O2 rebinding to Horse Heart Myoglobinafter photolysis (inside borate buffer (pH=8.42))
Temperature (K) τ A1 (%) kBA (1011 s -1)
276 5.4±0.4 31 0.57
286 5.5±0.3 30 0.54
296 5.4±0.4 28 0.52
306 5.9±0.5 24 0.41
316 6.0±0.5 23 0.38
158
10-1 100 101 102 103 104 105 106 107 108 109 10100.0
0.2
0.4
0.6
0.8
1.0
phase 1: τ1=5.35ps (28%)phase 2: τ2=41ns, β=0.72 (37%)phase 3: τ3=150μs (35%)
Kinetics of MbO2
0-3ps: Simulation with single exponential 3ps-13ns: with sychrolock system 10ns-20ms:with flash photolysis system
N (t
)
Time (ps)
Figure A1.9 O2 recombination kinetics to horse heart myoglobin in 0.1Mborate buffer at room temperature (T≈296K).
159
decades, shows that there are three separated recombination processes for oxygen
molecules rebinding back to iron atoms. The first geminate recombination process
completes within the first 20ps. There is another recombination starts around 1ns and
ends around 1us. The slowest rebinding process happens in the 100’s of microsecond.
The amplitudes of these three phases are 28%, 37%, 35%, respectively.
Figure A1.10: Glycerol concentration dependent kinetics of O2 recombination to horse
heart myoglobin at room temperature (T≈296K). High concentrated stock solution of
metMb is made in borate buffer (pH=8.42). Pure glycerol (purity>99%) and borate buffer
are added into the stock solution weighted by mass ratio to generate the diluted samples.
Final oxymyglobin samples for the ultrafast pump-probe experiment (<13ns) are prepared
according to Method 2. The colored solids are single-exponential fits of the data from 3ps
to 400ps. The dashed lines are back-extrapolations of the fitting functions to time zero.
All data are normalized to make N(t)=1 at time t=0. Fitting parameters are listed on Table
4.2. Kinetics data detected by the flash photolysis system of O2 recombination to horse
heart myoglobin with no glycerol content in the buffer are transplanted on the kinetics
trace of oxymyoglobin in the same buffer (no glycerol) to illustrate the complete behavior
of the slow geminate phase. For the complete rebinding kinetics (red squares), the
amplitudes of those three phases are 38%, 32% and 30%, repectively.
Figure A1.11: Temperature dependence of MbO2 recombination kinetics inside glycerol
mixture. The glycerol mixture is made with 80% (weight percentage) borate buffer
(pH=8.42) and 20% glycerol. Oxymyoglobin samples are made with Method 2. Colored
solid lines are single-exponential fittings of the data between 3ps to 400ps. The dashed
lines are back-extrapolations of the fitting functions to time zero. All data are normalized
160
10-1 100 101 102 103 104 105 106 107 108 109 10100.0
0.2
0.4
0.6
0.8
1.0
Glycerol-Dependent Kineticsof Horse Heart Oxymyoglobin
0-3ps: Simulation with single exponential3ps-13ns: with sychrolock system Glycerol weight percentage:
0% 20% 50% 90%
10ns-20ms:with flash photolysis system
N (t
)
Time (ps)
Figure A1.10 Glycerol concentration dependent kinetics of O2 rebinding tohorse heart myoglobin at room temperature (T≈296K).
161
Table A1.2 Glycerol Dependence of O2 rebinding to Horse Heart Myoglobinafter photolysis (T=296K)
Glycerol Pt(%) τ A1 (%) kBA (1011 s -1)
0 4.6±0.4 38 0.82
20 4.9±0.2 51 1.04
50 5.0±0.2 57 1.12
90 4.8±0.2 79 1.66
162
0.1 1 10 100 1000 100000.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Fittings: 0.32+0.68e-t/4.47
0.50+0.50e-t/5.36
0.69+0.31e-t/5.09Buffer: 20% glycerol / 80% borate (pH 8.42)
Temperature Dependenceof MbO2 inside glycerol mixture
313K 293K 275K
N (t
)
Time (ps)
Figure A1.11 Temperature dependence of MbO2 rebinding kinetics insideglycerol mixture.
163
to make N(0)=1 at time zero. For the fast rebinding phases, no temperature dependent
rate change is observed. However, the amplitude is dramatically affected by the
temperature, compared with that inside the pure borate buffer. On the other hand, the
buffer effect on the second decaying process is also exhibiting much bigger than that of
the pure borate. It is noticeable that at temperature 275K, the second phase is hardly
observable in this timing window. Either it is diminished or is delayed.
Figure A1.12: Experimental data of temperature dependent O2 rebinding kinetics to
mutant MbV68W in 0.1M borate buffer (pH=8.42). The protein sample is made with
Method 2. Absorption spectra before and after the experiment are shown on Figure 4.2.
All the kinetics data recorded are normalized at the maximum values. No temperature
dependent difference is observed within the noise variation. Two fitting procedures are
applied to the data. One is a single exponential (4.2ps) with a stretched exponential
(1.35ns, beta=0.8). The other applied is two –exponential fitting (4.6ps, 1.29ns). Both fit
the raw data set.
Figure A1.13: Kinetics of oxygen rebinding to V68W Mb differentiated by preparation
methods. The protein samples are prepared at room temperatures (T≈295K). The whole
kinetics is fitted using a single exponential plus a stretched-exponential decay. Response
between 0-~3ps is generated from back-extrapolation of fitting function.
Figure A1.14: Kinetics of oxygen rebinding to Horse Heart Myoglobin differentiated by
preparation methods. Experimental data from the maximum to 400ps are fitted with a
single-exponential decay. The early time responses are back extrapolated from the fitting
functions.
164
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
Temperature Dependenceof V68WMbO2borate buffer pH=8.42
276K 285K 295K 305K
Fittings: 1 stretched +1 single exponential
0.81e-t/4.15+0.50e-(t/1351)0.82
+0.02 2 exponentials
0.81e-t/4.56+0.45e-t/1288+0.04
Δ A
Time (ps)
Figure A1.12 Experimental data of temperature dependent O2 rebindingkinetics to mutant MbV68W in 0.1 borate buffer(pH=8.42).
165
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
Kinetics of V68W MbO2 differentiated by preparation methods
V68W MbO2 prepared by Method 1 V68W MbO2 prepared by Method 2
Fittings: 1 stretched +1 exponential 0.34e-t/5.15+0.60e-(t/1321)0.74
+0.06 0.57e-t/4.67+0.41e-(t/1276)0.78
+0.02
N
(t)
Time (ps)
Figure A1.13 Kinetics of O2 rebinding to V68W Mb differentiated bypreparation methods.
166
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
MbO2 prepared by Method 1 MbO2 prepared by Method 2
Fittings: Single exponentials 0.27e-t/5.35+0.73 0.46e-t/4.86+0.54
Kinetics of MbO2differentiated by preparation methods
N
(t)
Time (ps)
Figure A1.14 Kinetics of oxygen rebinding to horse heart myoglobindifferentiated by preparation methods.
167
Figure A1.15: Temperature dependent recombination kinetics of Horse Heart MbO2
prepared with Method 2. The sample is prepared within borate buffer (pH=8.42).
Recorded data are normalized at the peak values.
Figure A1.16: Temperature dependent recombination kinetics of mutant V68W MbO2
inside glycerol mixture. The glycerol mixture is made with 80% (weight percentage)
borate buffer (pH=8.42) and 20% glycerol. The protein sample is generated through
Method 2. A single exponential decay plus a stretched exponential decay is used to fit the
raw data. The fitting functions are used to extrapolate kinetic trace before 3ps. All data
are normalized to make N(t)=1 at time t=0.
Figure A1.17: Temperature dependent recombination kinetics of mutant L29W MbO2 in
0.1M borate buffer (pH=8.42) after photolysis. The protein sample is diluted directly to
an O2 bound stock solution. Absorption spectrum before and after the temperature
dependence experiments are compared in Figure 4.3. Data between 3ps and 400ps are
fitted with single exponential decay. Back-extrapolation is used to normalize the kinetics
to make N(t)=1 at time zero.
Figure A1.18: Relationship between solvent viscosity and O2 recombination kinetics to
horse heart myoglobin. All the protein samples are made with method 2. Solvent
viscosity values are those for water/glycerol mixtures.
168
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
315K 277K 295K 305K
MbO2Borate buffer pH=8.42
Temperature Dependenceof MbO
2 prepared by Method 2
Δ A
Time (ps)
Figure A1.15 Temperature dependent recombination kinetics of horse heartMbO2 prepared with Method 2.
169
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
295K 305K 315KFittings:
0.62e-t/4.62+0.36e-(t/1305)0.83
+0.01 0.48e-t/4.43+0.50e-(t/1241)0.79
+0.02 0.42e-t/5.10+0.57e-(t/1383)0.79
+0.01
Temperature Dependent Kineticsof V68WMbO2 inside80%borate buffer(pH=8.42)/20%glycerolPrepared by method 2
Δ A
Time (ps)
Figure A1.16 Temperature dependent recombination kinetics of mutantV68W MbO2 inside glycerol mixture.
170
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
Fittings: 0.68e-t/5.72+0.32 0.73e-t/6.63+0.27 0.89e-t/4.91+0.11
315K 295K 277K
Temperature DependentKinetics of L29W MbO2borate buffer, pH=8.42
N (t
)
Time (ps)
Figure A1.17 Temperature dependent recombination kinetics of mutantL29W MbO2 0.1M borate buffer after photolysis.
171
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
η=1.76 (293K, 20% glycerol) η=1.79 (277K, 0% glycerol)
η=1.01 (295K, 0% glycerol) η=1.07 (313K, 20% glycerol)
Viscosity Effect on MbO2
Δ
A
Time (ps)
Figure A1.18 Relationship between solvent viscosity and O2 recombinationkinetics to horse heart myoglobin.
172
Table A1.3 Viscosity of Aqueous Glycerol Solutions in Centipoises/mPa s
http://www.dow.com/glycerine/resources/table18.htm
173
Appendix 2 UV-Vis Absorption Spectral Studies of Ferrous Fe-
protoporphyrin IX in Different Monomeric Complexes
174
400 500 6000.00.10.20.30.40.50.60.70.80.91.01.11.21.3
392nm416nm428nm441nm
412nm
400nm FeIIPPIX-COin Kpi buffer with 1% CTABpH=7
Met FeIIIPPIX Deoxy FeIIPPIX (adding Na2S2O4) FeIIPPIX-CO (flushing CO gas)
O
.D.
Wavelength (nm)
Figure A2.1
175
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
400nm
434nm
419nm
2MeIm-FeIIPPIX-CO in Kpi buffer with 1% CTABpH=7
Met FeIIIPPIX Met 2MeIm-FeIIIPPIX (adding 2MeIm) Deoxy 2MeIm-FeIIPPIX (adding Na2S2O4) 2MeIm-FeIIPPIX-CO (flushing CO gas)
O.D
.
Wavelength (nm)
Figure A2.2
176
400 500 600 7000
1
2
400nm
418nm
pyridine-FeIIPPIX-CO in Kpi buffer with 1%CTABpH=7
Met FeIIIPPIX pyridine+Met FeIIIPPIX(adding pyridine)bis (py)2-FeIIPPIX (adding Na2S2O4) pyridine-FeIIPPIX-CO (flushing CO gas)
O.D
.
Wavelength (nm)
Figure A2.3
177
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
432nm
401nm
445nm
434nm
CN-FeIIPPIX-CO in Kpi buffer with 1% CTABpH=7
Met FeIIIPPIX Met CN-FeIIIPPIX(adding CN)CN-FeIIPPIX (adding Na2S2O4) CN-FeIIPPIX-CO(flushing CO gas)
O.D
.
Wavelength (nm)
Figure A2.4
178
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
392nm415nm427nm440nm
400nm
402nm
414nm
phenol-FeIIPPIX-COin Kpi buffer with 1% CTABpH=7
Met FeIIIPPIX Met phenol-FeIIIPPIX (adding phenol) Deoxy phenol-FeIIPPIX (adding Na2S2O4) phenol-FeIIPPIX-CO (flushing CO gas)
O.D
.
wavelength (nm)
Figure A2.5
179
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
445nm
431nm402nm
400nmFeIIPPIX-NO with CN- in the solutionKpi buffer with 1% CTABpH=7
Met FeIIIPPIX Met CN-FeIIIPPIX(adding CN) CN-FeIIPPIX (adding Na2S2O4) CN+FeIIPPIX-NO(flushing NO gas)
O.D
.
Wavelength (nm)
Figure A2.6
180
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
398nm
410nm
394nm
phenol-FeIIPPIX-COin 80% glycerol/20% water
Met FeIIIPPIX Met phenol-FeIIIPPIX (adding phenol) Deoxy phenol-FeIIPPIX (adding Na2S2O4) phenol-FeIIPPIX-CO (flushing CO gas)
O.D
.
Wavelength (nm)
Figure A2.7
181
400 500 600 7000.0
0.5
1.0
1.5
2.0
2.5
418nm
411nm
396FeIIPPIX-CO in 80% glycerolpH=12
Met FeIIIPPIX Deoxy FeIIPPIX (adding Na2S2O4) FeIIPPIX-CO (flushing CO gas)
O.D
.
Wavelength (nm)Figure A2.8
182
400 500 6000.0
0.5
1.0
1.5
Inside 80% glycerol: deoxy FeIIPPIX CO-FeIIPPIX
Inside 1% CTAB: deoxy FeIIPPIX CO-FeIIPPIX
435nm
424nm
O
.D.
Wavelength (nm)
Figure A2.9
183
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
buffer:3M NaOH with 1%CTAB
deoxy FeIIPPIX Met FeIIIPPIX
593561
440
O.D
.
wavelength (nm)
OH‐
Figure A2.10 Agree with Figure 1 of Gomez. et al. (JACS, 127,50,200517636). The 441nm peak in FeIIPPIX in CTAB (Figure A1.1) is assigned to theOH- bound species.
184
300 400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
397nm
589555
431
Buffer: 80% methanol/20% 3M NaOH
Met FeIII-PPIX Deoxy FeII-PPIX
O.D
.
Wavelength (nm)
OH‐
OH‐
Figure A2.11 See Figure 1C in Gomez et al. where 435nm species is seen in50% ethanol and assigned to (OH-)2 bound species. The peak at 431nm doesnot appear in Figure A1.1 and the (OH-)2 species is not assigned to any peak inFigure A1.1.
185
350 400 450 500 550 600 650 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
593561
440
587551-555
431
Buffer:3M NaOH with 1% CTAB
Deoxy FeII-PPIX Deoxy FeII-PPIX sitting for 5hrs Deoxy FeII-PPIX sitting for 20hrs
O.D
.
wavelength (nm)
OH‐OH‐
OH‐
Figure A2.12 Bis (OH-)2 species forms slowly (20hours) in 3M NaOH and 1%CTAB.
431nm 440nm
186
350 400 450 500 550 600 650 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
Buffer:3M NaOH with 1% CTAB
588555-558
431
409 FeII-PPIX-CO FeII-PPIX-CO sitting for 5hrs FeII-PPIX-CO after 30mins photolysis
O.D
.
wavelength (nm)
OH‐ OH‐
OH‐CO
Figure A2.13 Two samples: one sits for 5 hours and one is photolized using a403nm pump (2.0mW) and probed at 435nm (0.4mW). Both show evidence ofbis (OH-)2 species formation (Gomez. et al. JACS. 127, 50, 2005, 17634-17643). Longer waiting does not change ratio of peaks. The ligands (CO, OH-)and (OH-, OH-) are in equilibrium with CTAB so that CO is not the primaryligand after waiting long time.
431nm409nm
187
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
pH dependence of FeIIPPIX in 1% CTAB
pH 5.52 392 415 428 535 570pH 7.00 392 414 428 537 571pH 9.91 416 428 545 569
pH=9.91 pH=7 pH=5.52
O.D
. (no
rmal
ized
at 3
92nm
)
Wavelength (nm)
H2O
Figure A2.14 Gomez (Gomez. et al. JACS. 127, 50, 2005, 17634-17643)assigns 428 peak to H2O ligand. Test with pH, but [H2O]≈55M, so not muchof a change.
188
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
540
Buffer:80% methanol/ 20% water
563
416 Deoxy FeIIPPIX Met FeIIIPPIX
O.D
.
Wavelength (nm)
H2O
H2O
Figure A2.15 Agree with Gomez (Gomez. et al. JACS. 127, 50, 2005, 17634-17643) Figure 1C (a) where 419nm peak is seen in 50% ethanol. I surmise thatthis must be the (H2O)2 complex and the peak in Figure A1.15 correlates withthe 416 peak in Figure A1.1.
189
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
428412
Buffer: CHES with 1% CTABpH=9.91
569545
416 428 Deoxy FeIIPPIX 30min Deoxy FeIIPPIX 22min Deoxy FeIIPPIX 17min Deoxy FeIIPPIX 12min Deoxy FeIIPPIX 9min Deoxy FeIIPPIX 6min Deoxy FeIIPPIX 3min Deoxy FeIIPPIX 0min Met FeIIIPPIX
O.D
.
Wavelength (nm)
Figure A2.16 Like Figure A1.1 except species at 441nm (OH- bound, seeFigure A1.10) is diminished. (?Given high pH~10, would expect more of441nm species. This is contrary to expectation.? A consistent explanationmight be that , at high concentration of OH-, another OH- binds to 441nmspecies and the absorption peaks moves to 431nm, which superimposes to428nm peak)
190
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
570535-538
Buffer:MES with 1% CTABpH=5.52
428
412
392
428415 Deoxy FeIIPPIX 30min
Deoxy FeIIPPIX 25min Deoxy FeIIPPIX 20min Deoxy FeIIPPIX 15min Deoxy FeIIPPIX 10min Deoxy FeIIPPIX 5min Deoxy FeIIPPIX 0min Met FeIIIPPIX
O.D
.
Wavelength (nm)
Figure A2.17 Some 441nm species observed at pH5.5.
191
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
416nm
pH 12.53 416 563pH 7 416 563pH 5.29 417 565
pH dependence of FeIIPPIX in 80%methanol/20% water
pH=5.29 pH=7 pH=12.53
O.D
. (no
rmal
ized
at s
oret
pea
ks)
Wavelength (nm)
Figure A2.18
192
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2 396nm
392
565536-540
Buffer:80% methanol/ 20% waterpH=2.30
Deoxy FeIIPPIX Met FeIIIPPIX
O.D
.
Wavelength (nm)
Figure A2.19 Uncertain how Sodium Dithionite reacts at pH2-3. However,392nm Species is also observed in FeII-PPIX in CTAB (Figure A1.1). Ispeculate on the possibility that the species formed here may be related to the392nm species seen in Figure A1.1. I think this is a 5-coordinate Fe2+ form.
193
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2In 80% Methanol/Water MixturepH=7
416nm
398nm
408nm
Ferric Fe(III)PPIX Ferrous Fe(II)PPIX CO-Fe(II)PPIX
O.D
.
Wavelength (nm)
Figure A2.20 Compare with Figure A1.13 for 1% CTAB and 3M NaOH,which suggests OH- as ligand trans to CO. It is unclear why CO bindingwould select OH- vs H2O ligand. Compare with Figure A1.11, A1.8 and A1.9where H2O is presumably bound trans to CO and absorption is at 412nm(alpha/beta bands are also different).
194
400 500 600 7000.0
0.2
0.4
0.6
0.8
1.0
1.2
In 80% Methanol/Water MixturepH=7
Ferric Fe(III)PPIX Ferrous Fe(II)PPIX NO-Fe(II)PPIX
416nm
395nm
398nm
O.D
.
Wavelength(nm)
Figure A2.21
195
Appendix 3 How does CO rebind to FeII-PPIX?
196
100 101 102 103 104
0.0
0.2
0.4
0.6
0.8
1.0
Kpi buffer with 1% CTABpH=7
pump at 403nmprobe at 435nm
10 Tesla 0 Tesla
PPIXCO in Magnetic Field
ΔA (a
rbitr
ary
units
)
Time(ps)
Figure A3.1
197
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
Pump at 403nmProbe at 435nm
2MeIm-PPIX-COPPIX-COIn Kpi buffer with 1% CTAB
Δ A
(arb
itrar
y un
it)
Time (ps)
Figure A3.2
198
1 10 100 1000 10000
0.0
0.2
0.4
0.6
0.8
1.0
pump: 403nmprobe: 435nm
Ferrous Fe-PPIX in 3M NaOH (1% CTAB)pH>14
Data: DEOXYFEPPIX3_WModel: ExpDec1 Chi^2 = 0.00023R^2 = 0.99821 y0 0.03385 ±0.00225A1 1.42349 ±0.01406t1 4.81252 ±0.07918
Δ A
Time (ps)
Figure A3.3
199
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
Pump 403nmProbe 435nm
1-exponential fitting
2-exponential fitting
Data: PPIXCO435NM_WModel: ExpDec2 Chi^2 = 0.00002R^2 = 0.99654 y0 0.6978 ±0.00122A1 0.46331 ±0.01t1 4.18072 ±0.12638A2 0.04256 ±0.00233t2 250.79929 ±37.23482
PPIX-CO in 80% Methanol/water Mixture
Data: PPIXCO435NM_WModel: ExpDec1 Chi^2 = 0.0002R^2 = 0.97066 y0 0.71182 ±0.00223A1 0.44198 ±0.02084t1 5.38645 ±0.34437
Δ A
Time (ps)
Figure A3.4
200
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
Pump 403nmProbe 428nm
PPIX-CO in 80% Methanol/water Mixture
Δ A
Time (ps)
Figure A3.5
201
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
pH should be around 7
Pump 403nmProbe 428nm
2MeIm-PPIX-CO in 80% Methanol/water Mixture
ΔA
Time (ps)
Figure A3.6
202
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
Pump 403nmProbe 435nm
2MeIm-PPIX-CO in 80% Methanol/water Mixture
Δ A
Time (ps)
Figure A3.7
203
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
Pump at 403nmProbe at 435nm
2MeIm-PPIX-CO in glycerol-CTAB-buffer system1% CTAB
95% 80% 65% 50% 35% 20% 5% 0%
Δ A
(arb
itrar
y un
it)
Time (ps)
Figure A3.8
204
Figure A3.9
205
0.1 1 10 100 1000 100000.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Pump at 403nmProbe at 435nm
2MeIm-PPIX-CO in glycerol-water system
50% 65% 80%
Δ A
(arb
itrar
y un
it)
Time (ps)
Figure A3.10
206
Appendix 4 How does NO rebind to FeII-PPIX?
207
1E9 1E10 1E11 1E120
1
2
3
MEM analysis
Rate (s-1)
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
pump at 403nmprobe at 435nm
2MeIm-PPIX-NOPPIX-NOIn Kpi buffer with 1% CTAB
Time (ps)
ΔA
(arb
itrar
y un
its)
Figure A4.1
208
10-1 100 101 102 103 104 105 106 107 108 109 1010
1E-4
1E-3
0.01
0.1
1 PPIX-NO, 2MeImPPIX-NOIn Kpi buffer with 1% CTAB
Time (ps)
ΔA
(arb
itrar
y un
its)
NO85%
~ 10ps
15%~ 1 µs2MeIm NO~100 µs
Courtesy of Wenxiang ’s data
Figure A4.2 The whole kinetics of NO rebinding to FeIIPPIX, with and without0.1M 2‐MeIm inside the sample solutions. In both cases, 85% photolyzed NOrebinds back geminately with a time constant around 10ps. Without 2MeIminside the solution, the other 15% photolyzed NO will bind back bi‐molecularly later around ~100µs; With 2MeIm in the solution, ΔA will firstincrease at a time constant around 1µs then decay at time constant around100µs. In one of my speculations, this difference results from that the 2MeImrebinds bi‐molecularly to photolyzed FeII‐PPIX faster than NO molecular. Inthis scheme, NO binds from the other side of the 2MeIm‐bound FeII‐PPIX,and then, 2MeIm leaves due to tran effect.
209
1 10 100 1000 10000
0.0
0.2
0.4
0.6
0.8
1.0
Data: NO2MEIMPPIX_WModel: ExpDec2 Chi^2 = 0.00008R^2 = 0.99801 y0 0.30204 ±0.00181A1 0.13831 ±0.01115t1 81.27998 ±10.00317A2 0.83055 ±0.01341t2 6.6617 ±0.24549
Pump 403nmProbe 435nm
PPIX-NO in 80% Methanol/water Mixture
Δ A
Time (ps)
Figure A4.3
210
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
PPIX-NO in different media
Temperature: 296Kpump at 403nm, probe at 435nm
80% Glycerol+20% Kpi 80% Glycerol+20% Kpi+1% CTAB Kpi+1% CTAB 80% Methanol+20% water
Δ A
Time (ps)
Figure A4.4
211
0.1 1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
pump at 403nmprobe at 435nm
Kpi buffer with 1% CTABpH=7
Temperature Dependence of PPIX-NO
316K-5th 305K-4th 296K-1st 286K-2nd 275K-3rd 296K-6th
Δ A
Time (ps)
Figure A4.5
212
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
Pump:403nmProbe:435nm
275K 316K
Kpi buffer with 1% CTABpH=7
Temperature Dependence of PPIX-NOΔ A
Time (ps)
1E9 1E10 1E11 1E120
1
2
3
4
Figure A4.6
213
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
PPIX-NO20%Kpi buffer with 1% CTABin 80% GlycerolpH=7
296K-1st 316K-2nd 275K-3rd
Δ A
Time (ps)
Figure A4.7
214
1 10 100 10000.01
0.1
1
pump at 403nmprobe at 435nm
PPIX-NO in 80% glycerol
ΔA
Time (ps)
305K 295K 285K 275K
Figure A4.8
215
0.1 1 10 100 1000
0.01
0.1
1
PPIX-NO in glycerol at Room Temperature
Δ A
Time (ps)
35% Glycerol 50% Glycerol 65% Glycerol 95% Glycerol
Figure A4.9
216
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
1st 275K 2nd 284K 3rd 294K 4th 303K 5th 313K 6th 318K 7th 275K-again
PPIX-NO 80% methanol/water mixturepump:403nm; probe:435nm
Δ A
Time (ps)
Figure A4.10
217
Figure A4.11
1 10 100 1000 100000.0
0.2
0.4
0.6
0.8
1.0
313K
318K
275K
PPIX-NO 80% methanol/water mixturepump:403nm; probe:435nm
Δ
A
Time (ps)
109 1010 1011 1012
218
FeIIPPIX‐NO FeIIPPIX : NO
FeIIPPIX :: NO
FeIIPPIX + NO
hν
<10ps
<10ps~100ps
~100 µs
Figure A4.12
